Vaccine compositions and methods and uses thereof

Abstract

The present invention relates to vaccines and vaccine compositions, in particular vaccines and vaccine compositions inactivated with antibiotics or antibacterial compounds. In particular, the invention extends to a bacterial vaccine or vaccine composition wherein the bacteria has been inactivated by an antibiotic or an antibacterial compound. The invention further extends to the use of the vaccine and vaccine compositions as a therapeutic or as part of a vaccine in the prevention of infection with a bacterial infection, such as Bordetella pertussis, and the treatment and / or prophylaxis of bacterial diseases, such as pertussis disease (whooping cough).

Description

[0001] VACCINE COMPOSITIONS AND METHODS AND USES THEREOF

[0002] Field of the Invention

[0003] The present invention relates to vaccines and vaccine compositions, in particular vaccines and vaccine compositions inactivated with antibiotics or antibacterial compounds. In particular, the invention extends to a bacterial vaccine or vaccine composition wherein the bacteria has been inactivated by an antibiotic or an antibacterial compound. The invention further extends to the use of the vaccine and vaccine compositions as a therapeutic or as part of a vaccine in the prevention of infection with a bacterial infection, such as Bordetella pertussis, and the treatment and / or prophylaxis of bacterial diseases, such as pertussis disease (whooping cough).

[0004] Background to the Invention

[0005] Vaccines are the most effective medical intervention for preventing infectious diseases. Current approaches to vaccination include, live attenuated, inactivated, subunit, toxoid, viral vector and DNA or messenger RNA (mRNA) vaccines. Attenuated vaccines, made from weakened version of live viruses or bacteria are probably the most effective vaccine, but can be associated with risks from infection in immunocompromised individuals or because of reversion of attenuated to a virulent (disease causing) form of the pathogen. Inactivated vaccines are also effective but can be associated with side effects, including systemic inflammation. Subunit vaccines made from protein components of a virus or bacteria are generally safe, but are often not good at stimulating immune responses and require adjuvant that non-specifically boost immunity. Viral vector vaccines utilize modified versions of a virus as a vector to deliver proteins from other viruses or bacteria; their efficacy is limited by immune responses to the vector (anti-vector response), which can suppress the immune responses following booster vaccination. The DNA / mRNA vaccines are very versatile and easy to manufacture, but current evidence suggest that immune responses induced with mRNA vaccines do not persist because they are not good at inducing immunological memory. Most current vaccines are given by injection into the muscle or skin. A small number are given as nasal sprays (e.g. live attenuated influenza vaccines). Nasally-delivered vaccines are more effective than parenterally delivered vaccines at generating immune responses in the respiratory tract, especially in the nasal tract. Most infectious agents enter the body through mucosal membranes, and recent vaccine strategies have concentrated on the production of antibodies at these sites to block their entry. The stimulation of secretory immune responses, which includes mucosal IgA, the predominant antibody isotype in mucosal secretions with the capability to neutralise bacteria, bacterial products and viruses, is considered to be crucial to vaccine development. Currently most vaccines delivered by injection are not efficient at inducing a mucosal response.

[0006] Pertussis disease (whooping cough) is caused by the Gram-negative bacterium B. pertussis, which infects the upper and lower respiratory tract, causing considerable morbidity in children and adults and severe disease that can be fatal in infants [1] . Pertussis was a common paediatric infection until the whole-cell vaccines (wP) vaccine was introduced in the late 1940s and this vaccine played a key role in changing the global epidemiology of pertussis. In the United States, the annual incidence of pertussis dropped from more than 200,000 cases in the early 1930s to 1,010 cases in 1976 [2], The wP vaccines are suspensions ofthe entire B. pertussis organism that has been inactivated, usually with formalin. Although markedly reducing the risks from whooping cough, the wP vaccines were associated with side effects at the injection site and with serious systemic reactions, including whole-limb swelling, febrile seizures, persistent crying and very rarely, hypotonic-hyporesponsive episodes [3],

[0007] Concerns about side effects of wP vaccines led to the development of acellular pertussis (aP), based on 2-5 purified B. pertussis antigens, pertussis toxin (PT), filamentous haemagglutinin (FHA), pertactin (PRN), and fimbrial proteins 2 and 3 (FIM2 / 3) [4], With enhanced safety profiles, and despite being more expensive than wP vaccines, the aP vaccines were included in the paediatric immunization schedules of most high-income countries. However, the cheaper and more effective wP vaccines remain the vaccine of choice in low- and middleincome countries. From the late 1990s up until 2006 all European countries (except Poland) switched to aP vaccines. However, despite continuous high pertussis vaccine coverage in infants in most countries (>95%), the pathogen is still circulating and epidemic cycles occur every 2-5 years. Based on the European Centre for Disease Prevention and Control (ECDC) and World Health Organization (WHO) data [5, 6] an increase in the incidence of pertussis, especially in infants below one year of age, has been reported during the last decade in Europe and several industrialized countries [6], Although the aP vaccines have an improved safety profile and prevent severe pertussis, they are not as effective as the wP vaccine that they replaced. Current aP vaccines do not prevent nasal infection with B. pertussis, allowing transmission to non-immune individuals. These injected aP vaccines induce potent antibody responses that circulate in the blood (serum antibodies), but fail to induce local antibodies or T cells (immune cells of the cellular immune response) in the respiratory tract. Recent studies have suggested that T cells that are resident in the respiratory tract play a key role in sustained protective immunity to B. pertussis. Conversely, first generation wP vaccines (made from inactivated whole bacteria), are more effective than aP vaccines, but are associated with significant local and systemic reactions, including fever and, more rarely, febrile seizures in children.

[0008] Studies in animal models showed that aP vaccines fail to prevent nasal colonization and transmission with B. pertussis [7, 8], supporting the evidence of asymptomatic transmission of B. pertussis from fully vaccinated individuals [9], This has been linked with a failure of the aP vaccines to induce local antibodies and T cells in the respiratory tract [9-11], Borkner et al showed IL-17-producing pathogen-specific CD4 tissue-resident (TRM) cells accumulate in the nasal tissue during primary infection with B. pertussis and these TRM cells expanded locally following re-infection with B. pertussis and play a critical role in nasal clearance of B. pertussis

[0010] , Moreover, protection against lung infection is mediated by Thl

[0011] cells and antibodies

[0012] , Parenteral immunization with wP vaccines induces Thl, Thl7 and TRM responses [7, 13, 14] and circulating B. pertussis-specific IgG, but do not generate mucosal antibody responses

[0015] , In contrast, current parenterally delivered alum-adjuvanted aP vaccines, induce potent circulating serum IgG antibodies and Th2 -biased responses, but do not induce mucosal IgA, Thl, Thl7 or TRM cells in the respiratory tissue [10, 16],

[0009] Most natural infections with B. pertussis in adolescents and adults are mild or subclinical and are often not reported

[0017] , Berbers et al showed a very large proportion of middle-aged adults in 18 EU / EEA countries (which were mainly immunized with wP vaccines) have low or undetectable antibodies against PT (<50 lU / mL), and therefore could be susceptible to developing pertussis

[0018] , This study also demonstrates that despite well implemented childhood vaccination programmes, there is high circulation of B. pertussis and a significant number of unreported cases in the middle-aged population

[0018] . Consequently, young infants are at high risk for serious disease, through transmission from parents, siblings and other close family members. In addition to the problem of pertussis in infants too young to be immunized, there are increasing numbers of cases in preadolescents, adolescents, and adults, suggesting immunity wanes rapidly, even after five doses of immunization with aP vaccines

[0019] , Although booster vaccination may provide a solution, boosting with aP vaccine only provides optimal protection for up to 1 year, with protection substantially waning 2-3 years later

[0020] , Currently, the WHO has recommended that countries still using wP vaccines should not switch to aP vaccines. However, the reintroduction of wP vaccines is not a possibility in most middle to high income countries, because it is deemed to be unsafe.

[0010] The incidence of pertussis is increasing worldwide despite widespread vaccine coverage. The resurgence of pertussis has been attributed to a number of factors. The emergence of B. pertussis strains with deletions or mutations in PT and PRN, key protective antigens in the aP vaccine, may have enabled escape from protective immunity induced with aP vaccines [21, 22], The current aP vaccines do not confer complete protection against infection, especially in the nasal tract, allowing transmission to non-immune individuals. Furthermore, there is evidence that induction or recruitment of TRM cells to the respiratory tract is suppressed in mice immunized with current aP vaccines

[0023] , Alternatively recent studies have demonstrated that intranasal delivery of attenuated B. pertussis or aP vaccine with mucosal adjuvants can induce B. pertussis-specific IgG and IgA and Thl- and Thl7-type respiratory TRM cells [16, 24],

[0011] New approaches are necessary for providing improved vaccines. Specifically, new approaches are necessary for providing improved vaccines that induce immunological memory in the lungs and nasal cavity and consequently better long-term immunity. More particularly, there is a clinical need for new vaccine approaches that are safe and highly effective for inducing sterilizing immunity against bacterial infection in humans. In addition, vaccines are urgently needed that help prevent transmission of bacteria, such as B. pertussis, from immunized individuals to others. In particular, there is a clinical need for B. pertussis vaccines that are considerably more effective than the current aP vaccine and safer than the older wP vaccine. Summary of the Invention

[0012] The inventors of the present invention have discovered a new vaccine platform approach based on inactivating bacteria with antibiotics or antibacterial compounds. The inventors have developed a novel vaccine for use in the prevention of bacterial infections, methods of treatment and manufacture thereof.

[0013] By way of example only, the inventors have developed a novel vaccine platform for use in the prevention of infection with B. pertussis and prevention and / or the treatment and / or prophylaxis of pertussis disease, also known as whooping cough. In particular aspects, the vaccine approach involves the aerosol or nasal delivery of bacteria that has been inactivated by antibiotics or antibacterial compounds. Advantageously, the vaccines and vaccine compositions of the present invention confer long term sterilising immunity in the upper and lower respiratory tract. The present inventors have discovered the potential of using antibiotic-inactivated bacteria as an aerosol or intranasally-delivered mucosal vaccine against bacteria, particularly bacteria that infect the upper and lower respiratory tract, using B. pertussis as an example.

[0014] They have discovered that treatment of B. pertussis and other bacteria, such as S. aureus and E. coli, in culture with ciprofloxacin or levofloxacin, both fluoroquinolone antibiotics, inhibited bacterial cell division, did not result in bacterial lysis, and resulted in enlargement of the bacteria. These results were not observed with non-fluoroquinolone antibiotics, such as chloramphenicol, kanamycin, penicillin or streptomycin. They have also demonstrated that bacteria treated with fluoroquinolone, but not other antibiotics, are highly immunogenic, inducing potent antigen-specific Thl and Thl7 and TRM cells in the respiratory tract for example.

[0015] For example, they discovered that treatment with these fluoroquinolone antibiotics, including ciprofloxacin, completely inactivated B. pertussis in vitro and did not give rise to an infection of the nose or lungs when given to mice by aerosol administration. Furthermore, the aerosol-delivered antibiotic-inactivated B. pertussis (AIBP) vaccine did not promote systemic pro-inflammatory cytokine production when delivered by aerosol, whereas a wP vaccine delivered by injection induced high level of inflammatory cytokines in the serum. Moreover, pertussis toxin (PT), a key virulence factor of B. pertussis, and mediator of reactogenicity of the wP vaccine, was not detectable in the respiratory tract of mice following aerosol delivery of AIBP, but was produced following aerosol delivery of live B. pertussis. These findings demonstrate that the inactivated bacterial vaccines of the present invention have a better safety profile than a conventional inactivated bacterial vaccine administered parenterally.

[0016] The present inventors also surprisingly demonstrate that respiratory immunization of mice by exposure to an aerosol of ciprofloxacin-treated B. pertussis vaccine induces B. pertussisspecific Thl and Thl7-type CD4 TRM cells in the lung and nasal tissue and B. pertussis-specific IgGl, IgG2c and IgA responses in serum and nasal associated lymphoid tissue (NALT). Importantly, the vaccines of the present invention completely protect mice against lung and nasal infection with bacterial infections such as B. pertussis. Similar results have been shown using fluoroquinolone inactivated S. aureus which typically causes a wide range of infections and diseases, from localized skin infections to severe systemic diseases, including but not limited to: skin and soft tissue infections and / or diseases, including boils, abscesses, cellulitis, impetigo, folliculitis and staphylococcal scalded skin syndrome (SSSS); bloodborne / bloodstream infections (bacteraemia), including sepsis; endocarditis; pneumonia; toxic shock syndrome; bone and joint infections, including osteomyelitis and septic arthritis.

[0017] These diseases / infections can be difficult to treat when present as methicillin-resistant Staphylococcus aureus (MRSA). Vaccines for E. coli are also contemplated, for example to treat urinary tract infections (UTIs), pneumonia, meningitis, and severe invasive E. coli disease (IED), which can lead to sepsis and other bloodborne / bloodstream infections (bacteraemia).

[0018] These findings demonstrate that vaccines prepared by inactivation of gram-negative or gram-positive bacteria with fluoroquinolone (or quinolone) antibiotics, but not with other antibiotics, are highly immunogenic. Furthermore, the inventors found that inactivation of gram-negative or gram-positive bacteria with fluoroquinolone antibiotics did not lyse the bacteria and their morphology and antigen structure remained intact. The inventors postulate that this results in enhanced immunogenicity when compared to bacteria inactivated by other means, such as conventional heat treatment or chemical treatment (e.g. conventional aldehyde treatment). These unexpected findings provide an ideal new vaccine platform approach for the development of potent vaccines against a wide range of infectious diseases going well beyond the exemplified B. pertussis. Additionally, while the fluoroquinolone-treated bacteria tested are highly immunogenic when delivered by aerosol or intranasal routes, this vaccine platform approach could also be used to prepare vaccines against other infectious diseases at other mucosal surfaces, such as the gastrointestinal or urinaiy / reproductive tract, which could be facilitated by oral delivery of the vaccine or indeed, any other conventional mucosal delivery route. Furthermore, this vaccine platform approach may also be applicable for treatment of other infections, e.g. S. aureus skin infections or the like, by conventional parenteral routes, such as intramuscular (IM), subcutaneous (SC / SQ), intradermal (ID), transdermal (TD), intraperitoneal (IP) or intravenous (IV) routes. Typically, parenteral administration is by injection.

[0019] Furthermore, the vaccines of the present invention could initially be used as a booster vaccine against bacterial infections or additionally / alternatively as a primary vaccination against bacterial infections, such as pertussis, in adolescents and adults. For example, the AIBP vaccine described herein could also be used for primary vaccination of infants and has the advantage over current bacterial vaccines of inducing local immune responses in the respiratory tract that prevent infection of the nose as well as lungs. In particular, the AIBP vaccine described herein prevents infection of the nose as well as lungs, thereby stopping transmission and community spread of B. pertussis. Alternatively, a prime-boost approach involving parenteral primary immunization using conventional vaccines followed by respiratory booster immunization of the vaccine of the invention could be adopted. For example, for B. pertussis individuals already immunized with the aP vaccine by a parenteral route could be subsequently boosted with AIBP vaccine of the present invention given by aerosol or nasal route.

[0020] An additional advantage of the AIBP vaccine described herein is its ability to induce potent immunological memory in the respiratory tract, thereby conferring long-term sterilizing immunity against B. pertussis infection. Antibiotic treated bacteria and bacteria treated with an anti-bacterial compound, especially when delivered by mucosal routes (intranasal, aerosol, pulmonary or oral) have the capacity to be used as vaccines against a range of bacterial pathogens that infect mucosal surfaces, including the lungs, nasal cavity and gastrointestinal tract.

[0021] Accordingly, a general aspect of the present invention provides a vaccine composition comprising bacteria inactivated by an antibiotic or an antibacterial compound for use in the prevention of infection with the bacteria and / or in the treatment and / or prophylaxis of a disease associated with the bacteria.

[0022] According to a first aspect of the present invention, there is provided a vaccine composition comprising bacteria inactivated by a quinolone antibiotic, preferably a fluoroquinolone antibiotic, or an antibacterial compound.

[0023] Throughout this description, the antibiotic will be recited as a fluoroquinolone antibiotic, but it will be understood that a quinolone antibiotic may also be used. Furthermore, any antibiotic, not limited to a quinolone or fluroquinolone antibiotics, which targets essential bacterial enzymes involved in metabolic pathways, protein synthesis, DNA replication and repair, permeates bacterial membrane and / or kills the bacteria by arresting cell division by inhibiting the function of DNA gyrase and / or DNA Topoisomerase IV may be used in the present invention.

[0024] The bacteria inactivated by the fluoroquinolone antibiotic is not lysed. It retains an intact morphology with bacterial antigens intact, preferably, wherein at least one or more bacterial antigens are not modified. This provides for an unexpected and enhanced immunogenicity compared to bacteria inactivated by other means such as heat treatment or chemical (e.g. aldehyde) treatment.

[0025] In embodiments, the bacteria is a gram-negative bacteria, such as B. pertussis or E.coli, or a gram-positive bacteria, such as S. aureus, and including acid-fast bacteria such as Tuberculosis complex M. tuberculosis, M. bovis), Leprosy-causing M. leprae)., Opportunistic nontuberculous mycobacteria M. kansasii, M. avium).

[0026] The bacteria may cause respiratory tract infections or a disease of the respiratory tract. Alternatively, or additionally, the bacteria may cause infections or diseases in other organs, such as the gastrointestinal (GI) tract, urinary / reproductive tract or skin. For these reasons multiple delivery routes are contemplated and discussed below. For example, administration at a mucosal site may be optimal when targeting diseases of the respiratory, GI or urinary tract, with nasal or aerosol delivery of antibiotic inactivated Bordetella pertussis vaccine being discussed in detail below and in the Examples. Additionally, when targeting skin diseases (i.e. non-mucosal surfaces), administration by parenteral routes may be contemplated. For example, the antibiotic inactivated bacteria could be delivered by parenteral routes, such as intramuscular (IM), subcutaneous (SC / SQ), intradermal (ID), transdermal (TD), intraperitoneal (IP) or intravenous (IV) routes. Typically, parenteral administration is by injection. This parenteral route could be applicable for a vaccine against skin or other infections with Staphylococcus aureus.

[0027] In embodiments in which the bacteria causes respiratory tract infections or a disease of the respiratory tract, the bacteria is Bordetella pertussis, Staphylococcus aureus, Burkholderia pseudomallei, Chlamydophila pneumoniae, Corynebacterium diphtheriae, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, Streptococcus aureus, Streptococcus pyogenes, Escherichia Coli, Pseudomonas aeruginosa, Coxiella burnetiid, Legionella pneumophila, Mycobacterium tuberculosis or non-tuberculous mycobacteria.

[0028] Preferably, the bacteria is Bordetella pertussis. In embodiments, the bacteria is Bordetella pertussis. In embodiments, the disease associated with the bacteria is pertussis disease.

[0029] Advantageously, the vaccine composition comprising bacteria inactivated by the fluoroquinolone antibiotic provides a greater immune response, in terms of activating antigen presenting cells and inducing protective T cell responses in lung and nasal tissue compared to bacteria inactivated by heat treatment or chemical treatment.

[0030] Still advantageously, the vaccine composition comprising bacteria inactivated by the fluoroquinolone antibiotic provides a greater immune response in terms of inducing Thl7 response in lung and nasal tissue compared to bacteria inactivated by heat treatment or chemical treatment.

[0031] Examples 1 and 2 provide extensive data establishing that the AIBP vaccine is more effective than conventional wP vaccines or current aP in terms of inducing local T cell responses in the lung and nasal tissue and in inducing sterilizing immunity against lung and nasal infection with B. pertussis. For example, the AIBP vaccine induced DC maturation and production of the T cell polarizing cytokines IL-ip, IL-12p70 and IL-23, which was significantly greater than that induced with the wP vaccine. These findings demonstrating that vaccine compositions of the invention comprising bacteria inactivated with fluoroquinolone antibiotics, are unexpectedly highly immunogenic when delivered via the respiratory tract. For example, aerosol administration of B. pertussis or S. aureus treated with either ciprofloxacin or levofloxacin induced potent antigen-specific IFN-y and IL-17-secreting T cell responses in the respiratory tract. In contrast, immunization with B. pertussis treated with chloramphenicol did not generate B. pertussis specific T cell responses, suggesting that the inactivation with fluoroquinolone antibiotics creates highly immunogenic bacteria / vaccine. The present inventors postulate that the enhanced immunogenicity of bacteria treated with fluoroquinolone antibiotics compared with other antibiotic treatments reflects the difference in the structure of the bacteria observed post-antibiotic treatment, in that the antibiotic treated bacteria are not lysed with intact, and sometimes enlarged, morphology.

[0032] In embodiments, the antibiotic is a fluoroquinolone antibiotic. In preferred embodiments, the fluoroquinolone antibiotic is ciprofloxacin, gemifloxacin, levofloxacin, moxifloxacin, delafloxacin, norfloxacin or ofloxacin. In more preferred embodiments, the fluoroquinolone antibiotic is ciprofloxacin.

[0033] In embodiments, gene(s) expressing proteins and / or antibiotic that target essential bacterial enzymes involved in DNA replication and repair, are delivered to the bacteria using a vector. In embodiments, the vector is DNA, RNA or a viral vector with protein / or antibioticexpressing gene(s) cloned.

[0034] In embodiments, the antibacterial compound is an essential oil, lysozyme, a bacteriocin, an antiseptic, a disinfectant or a metal nanoparticle. In embodiments, the essential oil is selected from the group but not limited to tea tree oil or oregano oil. In embodiments, the antiseptic is selected from the group but not limited to alcohol, chlorhexidine and triclosan. In embodiments, the metal nanoparticle comprises silver or copper. In a similar manner to the antibiotic, the antibacterial composition of the invention may also be a composition which targets essential bacterial enzymes involved in metabolic pathways, protein synthesis, DNA replication and repair, permeates bacterial membrane and / or kills the bacteria by arresting cell division by inhibiting the function of DNA gyrase and / or DNA Topoisomerase IV. In embodiments, the vaccine composition prevents infection of the nose and lungs with the bacteria.

[0035] In embodiments, the vaccine composition is administered by aerosol delivery. In embodiments, aerosol delivery involves the use of aerosol delivery devices, such as, basic pressurized metered-dose inhalers, nebulizers, valved holder chambers inhalers, dry powder inhalers, soft mist inhalers, nasal spray devices, nasal sprayers with syringe, and smart inhalers.

[0036] In embodiments, one immunisation is administered by aerosol delivery per subject. In embodiments, two immunisations are administered by aerosol delivery per subject. In embodiments, two or more immunisations are administered by aerosol delivery per subject.

[0037] As discussed above, the vaccine of the invention may be used either as a primary immunization or as a booster immunization. For example, individuals already immunized with a conventional B. pertussis aP vaccine by a parenteral route could be boosted with AIBP vaccine of the invention given by aerosol or nasal routes or vice versa.

[0038] In embodiments, the vaccine composition is formulated for administration at a mucosal site. In embodiments, the vaccine composition is formulated for intranasal, pulmonary, oral, buccal, sublingual, perlingual, transcutaneous, gastrointestinal tract, rectal or vaginal administration. In preferred embodiments, the vaccine composition is formulated for intranasal administration. In embodiments, the vaccine composition may be formulated for administration ata mucosal site by inhalation, through spraying the vaccine composition (e.g. an aerosol formulation) into the nasal region.

[0039] In embodiments, the vaccine composition is administered mucosally. In embodiments, the vaccine composition is administered intranasally.

[0040] In embodiments, the vaccine composition is administered by aerosol delivery using a nebuliser from a culture at 1X105-1X109CFU / ml or by intranasal administration at a dose of lxlO5-lxlO9CFU in 0.1-1.0 ml PBS. In embodiments, one immunisation is administered per subject. In embodiments, two immunisations are administered per subject. In embodiments, two or more immunisations are administered per subject.

[0041] In embodiments, the vaccine composition comprises at least one antigen derived from the bacteria. In embodiments, the antigen is derived from an antibiotic-inactivated bacteria or a bacteria inactivated by an antibacterial compound.

[0042] In embodiments, the vaccine composition confers sterilising immunity in the upper and lower respiratory tract. In embodiments, the vaccine composition confers long term sterilising immunity in the respiratory tract. In embodiments, the vaccine composition confers long term sterilising immunity in the nasal tract.

[0043] In embodiments, the vaccine composition induces T cells that remain in the lung and nasal tissue as tissue-resident memory T (TRM) cells. In embodiments, the vaccine composition induces a Thl and / or Thl7-mediated immune response. In embodiments, the vaccine composition induces Bordetella pertussis-specific Thl and Thl7-type CD4 TRM cells in the lung and nasal tissue and / or B. pertussis-specific IgGl, IgG2c and IgA responses in serum and nasal associated lymphoid tissue (NALTj.

[0044] According to a second general aspect of the present invention, there is provided a method for preventing infection with bacteria and / or for the treatment and / or prophylaxis of a disease associated with the bacteria, said method comprising the step of:

[0045] (i) administering to a subject in need thereof a therapeutically effective amount of a vaccine composition comprising the bacteria inactivated with an antibiotic or an antibacterial compound.

[0046] According to a further aspect of the present invention, there is provided a method of mediating an immune response in a subject against a bacteria, said method comprising the steps of:

[0047] (i) providing a vaccine composition comprising the bacteria inactivated with an antibiotic or an antibacterial compound, and

[0048] (if) administering the vaccine composition to the subject in a therapeutically effective or prophylactically effective amount. According to a further aspect of the present invention, there is provided a composition comprising a bacteria inactivated with an antibiotic or an antibacterial compound for use in mediating an immune system in a subject in particular for use in preventing infection with the bacteria and / or in the treatment and / or prophylaxis of a disease associated with the bacteria.

[0049] In this manner, this second aspect of the invention provides a method of inducing an immune response in a subject against a bacteria, said method comprising the steps of:

[0050] (i) providing a vaccine composition comprising a bacteria inactivated with a fluoroquinolone antibiotic, and

[0051] (if) administering the vaccine composition to the subject in a therapeutically effective or prophylactically effective amount.

[0052] For example, there is provided a method for inducing an immune response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the vaccine composition of the invention.

[0053] According to this second aspect of the present invention, there is also provided a method for preventing infection with bacteria and / or for the treatment and / or prophylaxis of a disease associated with the bacteria, said method comprising the step of:

[0054] (i) administering to a subject in need thereof a therapeutically effective amount of a vaccine composition comprising the bacteria inactivated with a fluoroquinolone antibiotic.

[0055] In embodiments, the bacteria is a gram-negative bacteria, such as B. pertussis or E. coli, or a gram-positive bacteria, such as S. aureus, and including acid-fast bacteria such as Tuberculosis complex M. tuberculosis, M. bovis), Leprosy-causing M. leprae)., Opportunistic nontuberculous mycobacteria M. kansasii, M. avium).

[0056] The bacteria may cause respiratory tract infections or a disease of the respiratory tract. Alternatively, or additionally, the bacteria may cause infections or diseases in other organs, such as the GI tract, urinary / reproductive tract or skin. For these reasons multiple delivery routes are contemplated and discussed below. For example, administration at a mucosal site may be optimal when targeting diseases of the respiratory, GI or urinary tract, with nasal or aerosol delivery of antibiotic inactivated Bordetella pertussis vaccine being discussed in detail below and in the Examples. Additionally, when targeting skin diseases (i.e. non-mucosal surfaces), administration by parenteral routes may be contemplated. For example, the antibiotic inactivated bacteria could be delivered by parenteral routes, such as intramuscular (IM), subcutaneous (SC / SQ), intradermal (ID), transdermal (TD), intraperitoneal (IP) or intravenous (IV) routes. Typically, parenteral administration is by injection. This parenteral route could be applicable for a vaccine against skin or other infections with Staphylococcus aureus.

[0057] In embodiments, in which the bacteria causes respiratory tract infections or disease of the respiratory tract, the bacteria is Bordetella pertussis, Staphylococcus aureus, Burkholderia pseudomallei, Chlamydophila pneumoniae, Corynebacterium diphtheriae, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, Streptococcus aureus, Streptococcus pyogenes, Escherichia Coli, Pseudomonas aeruginosa, Coxiella burnetii or Legionella pneumophila, Mycobacterium tuberculosis or non-tuberculous mycobacteria..

[0058] Preferably, the bacteria is Bordetella pertussis. In embodiments, the bacteria is Bordetella pertussis. In embodiments, the disease associated with the bacteria is pertussis disease.

[0059] Advantageously, the vaccine composition comprising bacteria inactivated by the fluoroquinolone antibiotic provides a greater immune response, in terms of activating antigen presenting cells and inducing protective T cell responses in lung and nasal tissue compared to bacteria inactivated by heat treatment or chemical treatment.

[0060] Still advantageously, the vaccine composition comprising bacteria inactivated by the fluoroquinolone antibiotic provides a greater immune response in terms of inducing Thl7 response in lung and nasal tissue compared to bacteria inactivated by heat treatment or chemical treatment.

[0061] Examples 1 and 2 provide extensive data establishing that the AIBP vaccine is more effective than conventional wP vaccines or current aP in terms of inducing local T cell responses in the lung and nasal tissue and in inducing sterilizing immunity against lung and nasal infection with B. pertussis. For example, the AIBP vaccine induced DC maturation and production of the T cell polarizing cytokines IL-ip, IL-12p70 and IL-23, which was significantly greater than that induced with the wP vaccine. These findings demonstrating that vaccine compositions of the invention comprising bacteria inactivated with fluoroquinolone antibiotics, are unexpectedly highly immunogenic when delivered via the respiratory tract. For example, aerosol administration of B. pertussis or S. aureus treated with either ciprofloxacin or levofloxacin induced potent antigen-specific IFN-y and IL-17-secreting T cell responses in the respiratory tract. In contrast, immunization with B. pertussis treated with chloramphenicol did not generate B. pertussis specific T cell responses, suggesting that the inactivation with fluoroquinolone antibiotics creates highly immunogenic bacteria / vaccine. The present inventors postulate that the enhanced immunogenicity of bacteria treated with fluoroquinolone antibiotics compared with other antibiotic treatments reflects the difference in the structure of the bacteria observed post-antibiotic treatment, in that the antibiotic treated bacteria are not lysed with intact, and sometimes enlarged, morphology.

[0062] In embodiments, the antibiotic is a fluoroquinolone antibiotic. In preferred embodiments, the fluoroquinolone antibiotic is ciprofloxacin, gemifloxacin, levofloxacin, moxifloxacin, delafloxacin, norfloxacin or ofloxacin. In more preferred embodiments, the fluoroquinolone antibiotic is ciprofloxacin.

[0063] In embodiments, gene(s) expressing proteins and / or antibiotic that target essential bacterial enzymes involved in DNA replication and repair, are delivered to the bacteria using a vector. In embodiments, the vector is DNA, RNA or a viral vector with protein / or antibioticexpressing gene(s) cloned.

[0064] In embodiments, the antibacterial compound is an essential oil, lysozyme, a bacteriocin, an antiseptic, a disinfectant or a metal nanoparticle. In embodiments, the essential oil is selected from the group but not limited to tea tree oil or oregano oil. In embodiments, the antiseptic is selected from the group but not limited to alcohol, chlorhexidine and triclosan. In embodiments, the metal nanoparticle comprises silver or copper. In a similar manner to the antibiotic, the antibacterial composition of the invention may also be a composition which targets essential bacterial enzymes involved in metabolic pathways, protein synthesis, DNA replication and repair, permeates bacterial membrane and / or kills the bacteria by arresting cell division by inhibiting the function of DNA gyrase and / or DNA Topoisomerase IV. In embodiments, the vaccine composition prevents infection of the nose and lungs with the bacteria.

[0065] In embodiments, the vaccine composition is administered by aerosol delivery. In embodiments, aerosol delivery involves the use of aerosol delivery devices, such as, basic pressurized metered-dose inhalers, nebulizers, valved holder chambers inhalers, dry powder inhalers, soft mist inhalers, nasal spray devices, nasal sprayers with syringe, smart inhalers.

[0066] In embodiments, the method comprises administering one immunisation by aerosol delivery per subject. In embodiments, the method comprises administering two immunisations by aerosol delivery per subject. In embodiments, the method comprises administering two or more immunisations by aerosol delivery per subject. As discussed above the vaccine of the invention may be used either as a primary immunization or as a booster immunization. For example, individuals already immunized with a conventional B. pertussis aP vaccine by a parenteral route could be boosted with AIBP vaccine of the invention given by aerosol or nasal routes or vice versa.

[0067] In embodiments, the vaccine composition is formulated for administration at a mucosal site. In embodiments, the vaccine composition is formulated for intranasal, pulmonary, oral, buccal, sublingual, perlingual, transcutaneous, gastrointestinal tract, rectal or vaginal administration. In preferred embodiments, the vaccine composition is formulated for intranasal administration. In embodiments, the vaccine composition may be formulated for administration ata mucosal site by inhalation, through spraying the vaccine composition (e.g. an aerosol formulation) into the nasal region.

[0068] In embodiments, the method comprises administering the vaccine composition mucosally. In embodiments, the method comprises administering the vaccine composition intranasally.

[0069] In embodiments, the vaccine composition is administered by aerosol delivery using a nebuliser from a culture at 1X105-1X109CFU / ml or by intranasal administration at a dose of lxlO5-lxlO9CFU in 0.1-1.0 ml PBS. In embodiments, one immunisation is administered per subject. In embodiments, two immunisations are administered per subject. In embodiments, two or more immunisations are administered per subject.

[0070] In embodiments, the vaccine composition comprises at least one antigen derived from Bordetella pertussis. In embodiments, the antigen is derived from antibiotic-inactivated Bordetella pertussis.

[0071] In embodiments, the vaccine composition confers sterilising immunity in the upper and lower respiratory tract. In embodiments, the vaccine composition confers long term sterilising immunity in the respiratory tract. In embodiments, the vaccine composition confers long term sterilising immunity in the nasal tract.

[0072] In embodiments, the vaccine composition induces T cells that remain in the lung and nasal tissue as tissue-resident memory T (TRM) cells. In embodiments, the vaccine composition induces a Thl and / or Thl7-mediated immune response. In embodiments, the vaccine composition induces Bordetella pertussis-specific Thl and Thl7-type CD4 TRM cells in the lung and nasal tissue and / or B. pertussis-specific IgGl, IgG2c and IgA responses in serum and nasal associated lymphoid tissue.

[0073] According to a third general aspect of the present invention, there is provided a vaccine for use in vaccinating a subject against infection with a bacteria and / or a disease associated with the bacteria, wherein the vaccine comprises the bacteria inactivated with an antibiotic or an antibacterial compound.

[0074] According to this third aspect of the invention, there is provided a vaccine composition or vaccine of the invention, for use in inducing an immune response in a subject against the bacteria.

[0075] According to this third aspect of the invention, there is also provided a vaccine composition or vaccine of the invention, for use in preventing of infection with the bacteria and / or in the treatment and / or prophylaxis of a disease associated with the bacteria. In embodiments, the bacteria is a gram-negative bacteria, such as B. pertussis or E. coli, or a gram-positive bacteria, such as S. aureus, and including acid-fast bacteria such as Tuberculosis complex M. tuberculosis, M. bovis), Leprosy-causing M. leprae)., Opportunistic nontuberculous mycobacteria M. kansasii, M. avium).

[0076] The bacteria may cause respiratory tract infections or a disease of the respiratory tract. Alternatively, or additionally, the bacteria may cause infections or diseases in other organs, such as the GI tract, urinary / reproductive tract or skin. For these reasons multiple delivery routes are contemplated and discussed below. For example, administration at a mucosal site may be optimal when targeting diseases of the respiratory, GI or urinary tract, with nasal or aerosol delivery of antibiotic inactivated Bordetella pertussis vaccine being discussed in detail below and in the Examples. Additionally, when targeting skin diseases (i.e. non-mucosal surfaces), administration by parenteral routes may be contemplated. For example, the antibiotic inactivated bacteria could be delivered by parenteral routes, such as intramuscular (IM), subcutaneous (SC / SQ), intradermal (ID), transdermal (TD), intraperitoneal (IP) or intravenous (IV) routes. Typically, parenteral administration is by injection. This parenteral route could be applicable for a vaccine against skin or other infections with Staphylococcus aureus.

[0077] In embodiments, the bacteria may cause skin infections or a diseases of the skin, such as, but not limited to, skin infection with Staphylococcus aureus.

[0078] In embodiments in which the bacteria causes respiratory tract infections or a disease of the respiratory tract, the bacteria is Bordetella pertussis, Staphylococcus aureus, Burkholderia pseudomallei, Chlamydophila pneumoniae, Corynebacterium diphtheriae, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, Streptococcus aureus, Streptococcus pyogenes, Escherichia Coli, Pseudomonas aeruginosa, Coxiella burnetii or Legionella pneumophila, Mycobacterium tuberculosis or non-tuberculous mycobacteria.

[0079] Preferably, the bacteria is Bordetella pertussis. In embodiments, the bacteria is Bordetella pertussis. In embodiments, the disease associated with the bacteria is pertussis disease.

[0080] Advantageously, the vaccine composition comprising bacteria inactivated by the fluoroquinolone antibiotic provides a greater immune response, in terms of activating antigen presenting cells and inducing protective T cell responses in lung and nasal tissue compared to bacteria inactivated by heat treatment or chemical treatment.

[0081] Still advantageously, the vaccine composition comprising bacteria inactivated by the fluoroquinolone antibiotic provides a greater immune response in terms of inducing Thl7 response in lung and nasal tissue compared to bacteria inactivated by heat treatment or chemical treatment.

[0082] Examples 1 and 2 provide extensive data establishing that the AIBP vaccine is more effective than conventional wP vaccines or current aP in terms of inducing local T cell responses in the lung and nasal tissue and in inducing sterilizing immunity against lung and nasal infection with B. pertussis. For example, the AIBP vaccine induced DC maturation and production of the T cell polarizing cytokines IL-ip, IL-12p70 and IL-23, which was significantly greater than that induced with the wP vaccine. These findings demonstrating that vaccine compositions of the invention comprising bacteria inactivated with fluoroquinolone antibiotics, are unexpectedly highly immunogenic when delivered via the respiratory tract. For example, aerosol administration of B. pertussis or S. aureus treated with either ciprofloxacin or levofloxacin induced potent antigen-specific IFN-y and IL-17-secreting T cell responses in the respiratory tract. In contrast, immunization with B. pertussis treated with chloramphenicol did not generate B. pertussis specific T cell responses, suggesting that the inactivation with fluoroquinolone antibiotics creates highly immunogenic bacteria / vaccine. The present inventors postulate that the enhanced immunogenicity of bacteria treated with fluoroquinolone antibiotics compared with other antibiotic treatments reflects the difference in the structure of the bacteria observed post-antibiotic treatment, in that the antibiotic treated bacteria are not lysed with intact, and sometimes enlarged, morphology.

[0083] In embodiments, the vaccine composition prevents infection of the nose and lungs with the bacteria.

[0084] In embodiments, the vaccine is an aerosol delivered vaccine.

[0085] In embodiments, the vaccine is an intranasally delivered vaccine. In embodiments, the antibiotic is a fluoroquinolone antibiotic. In preferred embodiments, the fluoroquinolone antibiotic is ciprofloxacin, gemifloxacin, levofloxacin, moxifloxacin, delafloxacin, norfloxacin or ofloxacin. In more preferred embodiments, the fluoroquinolone antibiotic is ciprofloxacin.

[0086] In embodiments, gene(s) expressing proteins and / or antibiotic that target essential bacterial enzymes involved in DNA replication and repair, are delivered to the bacteria using a vector. In embodiments, the vector is DNA, RNA or a viral vector with protein / or antibioticexpressing gene(s) cloned.

[0087] In embodiments, the antibacterial compound is an essential oil, lysozyme, a bacteriocin, an antiseptic, a disinfectant or a metal nanoparticle. In embodiments, the essential oil is selected from the group but not limited to tea tree oil or oregano oil. In embodiments, the antiseptic is selected from the group but not limited to alcohol, chlorhexidine and triclosan. In embodiments, the metal nanoparticle comprises silver or copper. In a similar manner to the antibiotic, the antibacterial composition of the invention may also be a composition which targets essential bacterial enzymes involved in metabolic pathways, protein synthesis, DNA replication and repair, permeates bacterial membrane and / or kills the bacteria by arresting cell division by inhibiting the function of DNA gyrase and / or DNA Topoisomerase IV.

[0088] In embodiments, the vaccine is administered by aerosol delivery. In embodiments, aerosol delivery involves the use of aerosol delivery devices, such as, basic pressurized metered-dose inhalers, nebulizers, valved holder chambers inhalers, dry powder inhalers, soft mist inhalers, nasal spray devices, nasal sprayers with syringe, smart inhalers.

[0089] In embodiments, one immunisation is administered by aerosol delivery per subject. In embodiments, two immunisations are administered by aerosol delivery per subject. In embodiments, two or more immunisations are administered by aerosol delivery per subject.

[0090] As discussed above the vaccine of the invention may be used either as a primary immunization or as a booster immunization. For example, individuals already immunized with a conventional B. pertussis aP vaccine by a parenteral route could be boosted with AIBP vaccine of the invention given by aerosol or nasal routes or vice versa. In embodiments, the vaccine is formulated for administration at a mucosal site. In embodiments, the vaccine is formulated for intranasal, pulmonary, oral, buccal, sublingual, perlingual, transcutaneous, gastrointestinal tract, rectal or vaginal administration. In preferred embodiments, the vaccine is formulated for intranasal administration. In embodiments, the vaccine may be formulated for administration at a mucosal site by inhalation, through spraying the vaccine (e.g. an aerosol formulation) into the nasal region.

[0091] In embodiments, the vaccine is administered mucosally. In embodiments, the vaccine is administered intranasally.

[0092] In embodiments, the vaccine is administered by aerosol delivery using a nebuliser from a culture at 1X105-1X109CFU / ml or by intranasal administration at a dose of 1X105-1X109CFU in 0.1-1.0 ml PBS.

[0093] In embodiments, one vaccine is administered per subject. In embodiments, two vaccines are administered per subject. In embodiments, two or more vaccines are administered per subject.

[0094] In embodiments, the vaccine comprises at least one antigen derived from Bordetella pertussis. In embodiments, the antigen is derived from antibiotic-inactivated Bordetella pertussis.

[0095] In embodiments, the vaccine confers sterilising immunity in the upper and lower respiratory tract. In embodiments, the vaccine confers long term sterilising immunity in the respiratory tract. In embodiments, the vaccine confers long term sterilising immunity in the nasal tract.

[0096] In embodiments, the vaccine induces T cells that remain in the lung and nasal tissue as tissueresident memory T (TRM) cells. In embodiments, the vaccine induces a Thl and / or Thl7- mediated immune response. In embodiments, the vaccine induces Bordetella pertussisspecific Thl and Thl7-type CD4 TRM cells in the lung and nasal tissue and / or B. pertussisspecific IgGl, IgG2c and IgA responses in serum and nasal associated lymphoid tissue.

[0097] According to a fourth general aspect of the present invention, there is provided use of a vaccine composition comprising a bacteria inactivated with an antibiotic or an antibacterial compound in the preparation of a medicament for the treatment and / or prophylaxis of a disease associated with the bacteria.

[0098] In this manner, there is provided use of a bacteria inactivated with an antibiotic or an antibacterial compound in the preparation of a medicament for the treatment and / or prophylaxis of a disease associated with the bacteria.

[0099] In embodiments, the bacteria is a gram negative bacteria, such as B. pertussis or E. coli, or a gram-positive bacteria, such as S. aureus, and including acid-fast bacteria such as Tuberculosis complex M. tuberculosis, M. bovis), Leprosy-causing M. leprae)., Opportunistic nontuberculous mycobacteria M. kansasii, M. avium).

[0100] The bacteria may cause respiratory tract infections or a disease of the respiratory tract. Alternatively, or additionally, the bacteria may cause infections or diseases in other organs, such as the GI tract, urinary / reproductive tract or skin. For these reasons multiple delivery routes are contemplated and discussed below. For example, administration at a mucosal site may be optimal when targeting diseases of the respiratory, GI or urinary tract, with nasal or aerosol delivery of antibiotic inactivated Bordetella pertussis vaccine being discussed in detail below and in the Examples. Additionally, when targeting skin diseases (i.e. non-mucosal surfaces), administration by parenteral routes may be contemplated. For example, the antibiotic inactivated bacteria could be delivered by parenteral routes, such as intramuscular (IM), subcutaneous (SC / SQ), intradermal (ID), transdermal (TD), intraperitoneal (IP) or intravenous (IV) routes. Typically, parenteral administration is by injection. This parenteral route could be applicable for a vaccine against skin or other infections with Staphylococcus aureus.

[0101] In embodiments, in which the bacteria causes respiratory tract infections or a disease of the respiratory tract, the bacteria is Bordetella pertussis, Staphylococcus aureus, Burkholderia pseudomallei, Chlamydophila pneumoniae, Corynebacterium diphtheriae, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, Streptococcus aureus, Streptococcus pyogenes, Escherichia Coli, Pseudomonas aeruginosa, Coxiella burnetii or Legionella pneumophila, Mycobacterium tuberculosis or non-tuberculous mycobacteria. Preferably, the bacteria is Bordetella pertussis. In embodiments, the bacteria is Bordetella pertussis. In embodiments, the disease associated with the bacteria is pertussis disease.

[0102] Advantageously, the vaccine composition comprising bacteria inactivated by the fluoroquinolone antibiotic provides a greater immune response, in terms of activating antigen presenting cells and inducing protective T cell responses in lung and nasal tissue compared to bacteria inactivated by heat treatment or chemical treatment.

[0103] Still advantageously, the vaccine composition comprising bacteria inactivated by the fluoroquinolone antibiotic provides a greater immune response in terms of inducing Thl7 response in lung and nasal tissue compared to bacteria inactivated by heat treatment or chemical treatment.

[0104] Examples 1 and 2 provide extensive data establishing that the AIBP vaccine is more effective than conventional wP vaccines or current aP in terms of inducing local T cell responses in the lung and nasal tissue and in inducing sterilizing immunity against lung and nasal infection with B. pertussis. For example, the AIBP vaccine induced DC maturation and production of the T cell polarizing cytokines IL-ip, IL-12p70 and IL-23, which was significantly greater than that induced with the wP vaccine. These findings demonstrating that vaccine compositions of the invention comprising bacteria inactivated with fluoroquinolone antibiotics, are unexpectedly highly immunogenic when delivered via the respiratory tract. For example, aerosol administration of B. pertussis or S. aureus treated with either ciprofloxacin or levofloxacin induced potent antigen-specific IFN-y and IL-17-secreting T cell responses in the respiratory tract. In contrast, immunization with B. pertussis treated with chloramphenicol did not generate B. pertussis specific T cell responses, suggesting that the inactivation with fluoroquinolone antibiotics creates highly immunogenic bacteria / vaccine. The present inventors postulate that the enhanced immunogenicity of bacteria treated with fluoroquinolone antibiotics compared with other antibiotic treatments reflects the difference in the structure of the bacteria observed post-antibiotic treatment, in that the antibiotic treated bacteria are not lysed with intact, and sometimes enlarged, morphology.

[0105] In embodiments, the antibiotic is a fluoroquinolone antibiotic. In preferred embodiments, the fluoroquinolone antibiotic is ciprofloxacin, gemifloxacin, levofloxacin, moxifloxacin, delafloxacin, norfloxacin or ofloxacin. In more preferred embodiments, the fluoroquinolone antibiotic is ciprofloxacin.

[0106] In embodiments, gene(s) expressing protein and / or antibiotic that target essential bacterial enzymes involved in DNA replication and repair, are delivered to the bacteria using a vector. In embodiments, the vector is DNA, RNA or a viral vector with protein / or antibioticexpressing gene(s) cloned.

[0107] In embodiments, the antibacterial compound is an essential oil, lysozyme, a bacteriocin, an antiseptic, a disinfectant or a metal nanoparticle. In embodiments, the essential oil is selected from the group but not limited to tea tree oil or oregano oil. In embodiments, the antiseptic is selected from the group but not limited to alcohol, chlorhexidine and triclosan. In embodiments, the metal nanoparticle comprises silver or copper. In a similar manner to the antibiotic, the antibacterial composition of the invention may also be a composition which targets essential bacterial enzymes involved in metabolic pathways, protein synthesis, DNA replication and repair, permeates bacterial membrane and / or kills the bacteria by arresting cell division by inhibiting the function of DNA gyrase and / or DNA Topoisomerase IV.

[0108] In embodiments, the vaccine composition prevents infection of the nose and lungs with the bacteria.

[0109] In embodiments, the vaccine composition is administered by aerosol delivery. In embodiments, aerosol delivery involves the use of aerosol delivery devices, such as, basic pressurized metered-dose inhalers, nebulizers, valved holder chambers inhalers, dry powder inhalers, soft mist inhalers, nasal spray devices, nasal sprayers with syringe, smart inhalers.

[0110] In embodiments, one immunisation is administered by aerosol delivery per subject. In embodiments, two immunisations are administered by aerosol delivery per subject. In embodiments, two or more immunisations are administered by aerosol delivery per subject.

[0111] As discussed above the vaccine of the invention may be used either as a primary immunization or as a booster immunization. For example, individuals already immunized with a conventional B. pertussis aP vaccine by a parenteral route could be boosted with AIBP vaccine of the invention given by aerosol or nasal routes or vice versa. In embodiments, the vaccine composition is formulated for administration at a mucosal site. In embodiments, the vaccine composition is formulated for intranasal, pulmonary, oral, buccal, sublingual, perlingual, transcutaneous, gastrointestinal tract, rectal or vaginal administration. In preferred embodiments, the vaccine composition is formulated for intranasal administration. In embodiments, the vaccine composition may be formulated for administration ata mucosal site by inhalation, through spraying the vaccine composition (e.g. an aerosol formulation) into the nasal region.

[0112] In embodiments, the vaccine composition is administered mucosally. In embodiments, the vaccine composition is administered intranasally.

[0113] In embodiments, the vaccine composition is administered by aerosol delivery using a nebuliser from a culture at 1X105-1X109CFU / ml or by intranasal administration at a dose of lxlO5-lxlO9CFU in 0.1-1.0 ml PBS.

[0114] In embodiments, one immunisation is administered per subject. In embodiments, two immunisations are administered per subject. In embodiments, two or more immunisations are administered per subject.

[0115] In embodiments, the vaccine composition comprises at least one antigen derived from Bordetella pertussis. In embodiments, the antigen is derived from antibiotic-inactivated Bordetella pertussis.

[0116] In embodiments, the vaccine composition confers sterilising immunity in the upper and lower respiratory tract. In embodiments, the vaccine composition confers long term sterilising immunity in the respiratory tract. In embodiments, the vaccine composition confers long term sterilising immunity in the nasal tract.

[0117] In embodiments, the vaccine composition induces T cells that remain in the lung and nasal tissue as tissue-resident memory T (TRM) cells. In embodiments, the vaccine composition induces a Thl and / or Thl7-mediated immune response. In embodiments, the vaccine composition induces Bordetella pertussis-specific Thl and Thl7-type CD4 TRM cells in the lung and nasal tissue and / or B. pertussis-specific IgGl, IgG2c and IgA responses in serum and nasal associated lymphoid tissue.

[0118] According to a fifth general aspect of the present invention, there is provided an aerosol- delivered antibiotic-inactivated Bordetella pertussis (Al BP) vaccine or a Bordetella pertussis vaccine wherein the Bordetella pertussis has been inactivated with an antibacterial compound for use in preventing infection with Bordetella pertussis and / or in the treatment and / or prophylaxis of pertussis disease.

[0119] According to this fifth aspect of the invention, there is provided an aerosol-delivered fluoroquinolone antibiotic-inactivated vaccine. The bacteria may be B. pertussis or S. aureus. In embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBP) vaccine prevents infection of the nose and lungs with B. pertussis.

[0120] Advantageously, there is provided an aerosol-delivered fluoroquinolone antibiotic- inactivated Bordetella pertussis (AIBP) vaccine, wherein the vaccine prevents infection of the nose and lungs with B. pertussis.

[0121] In embodiments, the antibiotic is a fluoroquinolone. In preferred embodiments, the fluoroquinolone antibiotic is ciprofloxacin, gemifloxacin, levofloxacin, moxifloxacin, delafloxacin, norfloxacin or ofloxacin. In more preferred embodiments, the fluoroquinolone antibiotic is ciprofloxacin.

[0122] In embodiments, gene(s) expressing proteins and / or antibiotic that target essential bacterial enzymes involved in DNA replication and repair, are delivered to the bacteria using a vector. In embodiments, the vector is DNA, RNA or a viral vector with protein / or antibioticexpressing gene(s) cloned.

[0123] In embodiments, the antibacterial compound is an essential oil, lysozyme, a bacteriocin, an antiseptic, a disinfectant or a metal nanoparticle. In embodiments, the essential oil is selected from the group but not limited to tea tree oil or oregano oil. In embodiments, the antiseptic is selected from the group but not limited to alcohol, chlorhexidine and triclosan. In embodiments, the metal nanoparticle comprises silver or copper. In a similar manner to the antibiotic, the antibacterial composition of the invention may also be a composition which targets essential bacterial enzymes involved in metabolic pathways, protein synthesis, DNA replication and repair, permeates bacterial membrane and / or kills the bacteria by arresting cell division by inhibiting the function of DNA gyrase and / or DNA Topoisomerase IV.

[0124] In embodiments, aerosol delivery involves the use of aerosol delivery devices, such as, basic pressurized metered-dose inhalers, nebulizers, valved holder chambers inhalers, dry powder inhalers, soft mist inhalers, nasal spray devices, nasal sprayers with syringe, smart inhalers.

[0125] In embodiments, one immunisation is administered by aerosol delivery per subject. In embodiments, two immunisations are administered by aerosol delivery per subject. In embodiments, two or more immunisations are administered by aerosol delivery per subject. As discussed above the vaccine of the invention may be used either as a primary immunization or as a booster immunization. For example, individuals already immunized with a conventional B. pertussis aP vaccine by a parenteral route could be boosted with AIBP vaccine of the invention given by aerosol or nasal routes or vice versa.

[0126] In embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBP) vaccine is formulated for administration at a mucosal site. In embodiments, the aerosol- delivered antibiotic-inactivated Bordetella pertussis (AIBP) vaccine is formulated for intranasal, pulmonary, oral, buccal, sublingual, perlingual, transcutaneous, gastrointestinal tract, rectal or vaginal administration. In preferred embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBP) vaccine is formulated for intranasal administration. In embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBP) vaccine may be formulated for administration at a mucosal site by inhalation, through spraying the vaccine composition (e.g. an aerosol formulation) into the nasal region.

[0127] In embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBP) vaccine is administered mucosally. In embodiments, the aerosol-delivered antibiotic- inactivated Bordetella pertussis (AIBP) vaccine is administered intranasally.

[0128] In embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBP) vaccine is an intranasally delivered vaccine. In embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBP) vaccine is administered by aerosol delivery using a nebuliser from a culture at 1X105-1X109CFU / ml or by intranasal administration at a dose of 1X105-1X109CFU in 0.1-1.0 ml PBS.

[0129] In embodiments, one immunisation is administered per subject. In embodiments, two immunisations are administered per subject. In embodiments, two or more immunisations are administered per subject.

[0130] In embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBP) vaccine comprises at least one antigen derived from Bordetella pertussis. In embodiments, the antigen is derived from antibiotic-inactivated Bordetella pertussis.

[0131] In embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBPj vaccine confers sterilising immunity in the upper and lower respiratory tract. In embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBPj vaccine confers long term sterilising immunity in the respiratory tract. In embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBPj vaccine confers longterm sterilising immunity in the nasal tract.

[0132] In embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBPj vaccine induces T cells that remain in the lung and nasal tissue as tissue-resident memory T (TRM) cells. In embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBPj vaccine induces a Thl and / or Thl7-mediated immune response. In embodiments, the aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBPj vaccine induces Bordetella pertussis-specific Thl and Thl7-type CD4 TRM cells in the lung and nasal tissue and / or B. pertussis-specific IgGl, IgG2c and IgA responses in serum and nasal associated lymphoid tissue.

[0133] According to a further aspect of the present invention, there is provided a vaccine composition comprising Bordetella pertussis inactivated with an antibiotic or an antibacterial compound for use in the prevention of infection with Bordetella pertussis and / or in the treatment and / or prophylaxis of pertussis disease. All other features and limitations to the invention discussed in relation to previous aspects of the inventions will be applicable to this further aspect of the invention.

[0134] According to a further aspect of the present invention, there is provided a pharmaceutical composition for preventing infection with a bacteria and / or for the treatment and / or prophylaxis of a disease associated with the bacteria, wherein the pharmaceutical composition comprises an antibiotic-inactivated bacteria vaccine for preventing infection with the bacteria and / or for the treatment and / or prophylaxis of a disease associated with the bacteria, along with a pharmaceutically acceptable excipient, diluent or carrier.

[0135] All other features and limitations to the invention discussed in relation to previous aspects of the inventions will be applicable to this further aspect of the invention.

[0136] According to a further aspect of the present invention, the present invention extends to the vaccine of the invention, or to preparations or mixtures comprising the same, or to compositions containing the same, for use as a booster to enhance the immune response generated in a host against a bacteria to which the subject has been exposed, typically by way of infection or due to the previous administration of a primary vaccine. In this manner, the vaccine or vaccine composition of the invention may be used as a primary vaccine or as a booster vaccine.

[0137] All other features and limitations to the invention discussed in relation to previous aspects of the inventions will be applicable to this further aspect of the invention.

[0138] According to a further aspect of the present invention, the present invention further provides the vaccine composition of the invention for use in a method of vaccinating a subject to induce immunity against a bacteria.

[0139] All other features and limitations to the invention discussed in relation to previous aspects of the inventions will be applicable to this further aspect of the invention.

[0140] According to a further aspect of the present invention, there is provided a method of vaccinating a subject against infection from a bacteria and / or a disease associated with the bacteria, comprising administering an effective amount of a vaccine comprising the bacteria inactivated with an antibiotic or an antibacterial compound.

[0141] All other features and limitations to the invention discussed in relation to previous aspects of the inventions will be applicable to this further aspect of the invention.

[0142] In embodiments of these aspects of the present invention, the bacteria is a gram-negative bacteria, such as B. pertussis or E. coli, or a gram-positive bacteria, such as S. aureus and including acid-fast bacteria such as Tuberculosis complex M. tuberculosis, M. bovis), Leprosycausing M. leprae)., Opportunistic nontub erculous mycobacteria M. kansasii, M. avium).

[0143] The bacteria may cause respiratory tract infections or a disease of the respiratory tract. Alternatively, or additionally, the bacteria may cause infections or diseases in other organs, such as the GI tract, urinary / reproductive tract or skin. For these reasons multiple delivery routes are contemplated and discussed below. For example, administration at a mucosal site may be optimal when targeting diseases of the respiratory, GI or urinary tract, with nasal or aerosol delivery of antibiotic inactivated Bordetella pertussis vaccine being discussed in detail below and in the Examples. Additionally, when targeting skin diseases (i.e. non-mucosal surfaces), administration by parenteral routes may be contemplated. For example, the antibiotic inactivated bacteria could be delivered by parenteral routes, such as intramuscular (IM), subcutaneous (SC / SQ), intradermal (ID), transdermal (TD), intraperitoneal (IP) or intravenous (IV) routes. Typically, parenteral administration is by injection. This parenteral route could be applicable for a vaccine against skin or other infections with Staphylococcus aureus.

[0144] In embodiments of the aspects of the present invention, in which the bacteria causes respiratory tract infections or a disease of the respiratory tract, the bacteria is Bordetella pertussis, Staphylococcus aureus, Burkholderia pseudomallei, Chlamydophila pneumoniae, Corynebacterium diphtheriae, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, Streptococcus aureus, Streptococcus pyogenes, Escherichia Coli, Pseudomonas aeruginosa, Coxiella burnetii or Legionella pneumophila or Mycobacterium tuberculosis and non-tuberculous mycobacteria. Preferably, the bacteria is Bordetella pertussis. In embodiments of the aspects of the present invention, the bacteria is Bordetella pertussis. In embodiments, the disease associated with the bacteria is pertussis disease.

[0145] Advantageously, the vaccine composition comprising bacteria inactivated by the fluoroquinolone antibiotic provides a greater immune response, in terms of activating antigen presenting cells and inducing protective T cell responses in lung and nasal tissue compared to bacteria inactivated by heat treatment or chemical treatment.

[0146] Still advantageously, the vaccine composition comprising bacteria inactivated by the fluoroquinolone antibiotic provides a greater immune response in terms of inducing Thl7 response in lung and nasal tissue compared to bacteria inactivated by heat treatment or chemical treatment.

[0147] Examples 1 and 2 provide extensive data establishing that the AIBP vaccine is more effective than conventional wP vaccines or current aP in terms of inducing local T cell responses in the lung and nasal tissue and in inducing sterilizing immunity against lung and nasal infection with B. pertussis. For example, the AIBP vaccine induced DC maturation and production of the T cell polarizing cytokines IL-ip, IL-12p70 and IL-23, which was significantly greater than that induced with the wP vaccine. These findings demonstrating that vaccine compositions of the invention comprising bacteria inactivated with fluoroquinolone antibiotics, are unexpectedly highly immunogenic when delivered via the respiratory tract. For example, aerosol administration of B. pertussis or S. aureus treated with either ciprofloxacin or levofloxacin induced potent antigen-specific IFN-y and IL-17-secreting T cell responses in the respiratory tract. In contrast, immunization with B. pertussis treated with chloramphenicol did not generate B. pertussis specific T cell responses, suggesting that the inactivation with fluoroquinolone antibiotics creates highly immunogenic bacteria / vaccine. The present inventors postulate that the enhanced immunogenicity of bacteria treated with fluoroquinolone antibiotics compared with other antibiotic treatments reflects the difference in the structure of the bacteria observed post-antibiotic treatment, in that the antibiotic treated bacteria are not lysed with intact, and sometimes enlarged, morphology.

[0148] In embodiments of the aspects of the present invention, the antibiotic is a fluoroquinolone antibiotic. In preferred embodiments of the aspects of the present invention, the fluoroquinolone antibiotic is ciprofloxacin, gemifloxacin, levofloxacin, moxifloxacin, delafloxacin, norfloxacin or ofloxacin. In more preferred embodiments of the aspects of the present invention, the fluoroquinolone antibiotic is ciprofloxacin.

[0149] In embodiments, gene(s) expressing protein and / or antibiotic that target essential bacterial enzymes involved in DNA replication and repair, are delivered to the bacteria using a vector. In embodiments, the vector is DNA, RNA or a viral vector with protein / or antibioticexpressing gene(s) cloned.

[0150] In embodiments of the aspects of the present invention, the antibacterial compound is an essential oil, lysozyme, a bacteriocin, an antiseptic, a disinfectant or a metal nanoparticle. In embodiments of the aspects of the present invention, the essential oil is selected from the group but not limited to tea tree oil or oregano oil. In embodiments of the aspects of the present invention, the antiseptic is selected from the group but not limited to alcohol, chlorhexidine and triclosan. In embodiments of the aspects of the present invention, the metal nanoparticle comprises silver or copper. In a similar manner to the antibiotic, the antibacterial composition of the invention may also be a composition which targets essential bacterial enzymes involved in metabolic pathways, protein synthesis, DNA replication and repair, permeates bacterial membrane and / or kills the bacteria by arresting cell division by inhibiting the function of DNA gyrase and / or DNA Topoisomerase IV.

[0151] In embodiments, the vaccine composition prevents infection of the nose and lungs with the bacteria.

[0152] In embodiments of the aspects of the present invention, the vaccine or vaccine composition is administered by aerosol delivery. In embodiments of the aspects of the present invention, aerosol delivery involves the use of aerosol delivery devices, such as, basic pressurized metered-dose inhalers, nebulizers, valved holder chambers inhalers, dry powder inhalers, soft mist inhalers, nasal spray devices, nasal sprayers with syringe, smart inhalers.

[0153] In embodiments of the aspects of the present invention, one immunisation is administered by aerosol delivery per subject. In embodiments of the aspects of the present invention, two immunisations are administered by aerosol delivery per subject. In embodiments of the aspects of the present invention, two or more immunisations are administered by aerosol delivery per subject. As discussed above the vaccine of the invention may be used either as a primary immunization or as a booster immunization. For example, individuals already immunized with a conventional B. pertussis aP vaccine by a parenteral route could be boosted with AIBP vaccine of the invention given by aerosol or nasal routes or vice versa.

[0154] In embodiments of the aspects of the present invention, the vaccine or vaccine composition is formulated for administration at a mucosal site. In embodiments of the aspects of the present invention, the vaccine or vaccine composition is formulated for intranasal, pulmonary, oral, buccal, sublingual, perlingual, transcutaneous, gastrointestinal tract, rectal or vaginal administration. In preferred embodiments of the aspects of the present invention, the vaccine or vaccine composition is formulated for intranasal administration. In embodiments of the aspects of the present invention, the vaccine or vaccine composition may be formulated for administration at a mucosal site by inhalation, through spraying the vaccine composition (e.g. an aerosol formulation) into the nasal region.

[0155] In embodiments of the aspects of the present invention, the vaccine or vaccine composition is administered mucosally. In embodiments of the aspects of the present invention, the vaccine or vaccine composition is administered intranasally.

[0156] In embodiments of the aspects of the present invention, the vaccine composition is administered by aerosol delivery using a nebuliser from a culture at 1X105-1X109CFU / ml or by intranasal administration at a dose of 1X105-1X109CFU in 0.1-1.0 ml PBS.

[0157] In embodiments of the aspects of the present invention, one immunisation is administered per subject. In embodiments of the aspects of the present invention, two immunisations are administered per subject. In embodiments of the aspects of the present invention, two or more immunisations are administered per subject.

[0158] In embodiments, the vaccine or vaccine composition comprises at least one antigen derived from Bordetella pertussis. In embodiments of the aspects of the present invention, the antigen is derived from antibiotic-inactivated Bordetella pertussis.

[0159] In embodiments of the aspects of the present invention, the vaccine or vaccine composition confers sterilising immunity in the upper and lower respiratory tract. In embodiments, the vaccine or vaccine composition confers long term sterilising immunity in the respiratory tract. In embodiments of the aspects of the present invention, the vaccine or vaccine composition confers long term sterilising immunity in the nasal tract.

[0160] In embodiments of the aspects of the present invention, the vaccine or vaccine composition induces T cells that remain in the lung and nasal tissue as tissue-resident memory T (TRM) cells. In embodiments, the vaccine or vaccine composition induces a Thl and / or Thl7- mediated immune response. In embodiments of the aspects of the present invention, the vaccine or vaccine composition induces Bordetella pertussis-specific Thl and Thl7-type CD4 TRM cells in the lung and nasal tissue and / or B. pertussis-specific IgGl, IgG2c and IgA responses in serum and nasal associated lymphoid tissue.

[0161] According to another general aspect of the invention, there is provided a method for making a fluoroquinolone antibiotic inactivated bacterial vaccine, comprising:

[0162] (a) culturing bacteria from frozen stocks or clinical isolates;

[0163] (b) treating the cultured bacteria from (a) with an effective amount of fluoroquinolone antibiotic over an effective time period such that no live bacterial colony forming units (CFU) are present; and

[0164] (c) washing the treated bacteria from (b) free of fluoroquinolone antibiotic to result in a fluoroquinolone inactivated bacterial vaccine.

[0165] According to another general aspect of the invention, there is provided a method for inactivating a B. pertussis isolate, comprising:

[0166] (a) culturing B. pertussis bacteria from frozen stocks of B. pertussis Tohama-1 derivatives or from B. pertussis clinical isolates;

[0167] (b) treating the cultured B. pertussis from (a) with an effective amount of a fluoroquinolone antibiotic over a time period of approximately 1 to 24 hours such that no live B. pertussis CFU are present; and

[0168] (c) washing the treated B. pertussis from (b) free of fluoroquinolone antibiotic.

[0169] According to another general aspect of the invention, there is provided a method for making a fluoroquinolone antibiotic inactivated B. pertussis (AIBP) vaccine, comprising:

[0170] (a) culturing B. pertussis bacteria from frozen stocks of B. pertussis Tohama-1 derivatives or B. pertussis clinical isolates; (b) treating the cultured B. pertussis from (a) with an effective amount of fluoroquinolone antibiotic over a time period of approximately 1 to 24 hours such that no live B. pertussis CFU are present; and

[0171] (c) washing the treated B. pertussis from (b) free of the fluoroquinolone antibiotic to result in a fluoroquinolone inactivated B. pertussis vaccine.

[0172] According to another general aspect of the invention, there is provided a method for making a fluoroquinolone antibiotic inactivated S. aureus or other bacterial vaccine, comprising:

[0173] (a) culturing S. aureus bacteria from frozen stocks or S. aureus clinical isolates;

[0174] (b) treating the cultured S. aureus from (a) with an effective amount of fluoroquinolone antibiotic over a time period of approximately 1 to 24 hours such that no live S. aureus CFU are present; and

[0175] (c) washing the treated S. aureus from (b) free of the fluoroquinolone antibiotic to result in a fluoroquinolone inactivated S. aureus vaccine.

[0176] According to another general aspect of the invention, there is provided a method for making a fluoroquinolone antibiotic inactivated E. coli or other bacterial vaccine, comprising:

[0177] (a) culturing E. coli bacteria from frozen stocks or S. aureus clinical isolates;

[0178] (b) treating the cultured E. coli from (a) with an effective amount of fluoroquinolone antibiotic over a time period of approximately 1 to 24 hours such that no live S. aureus CFU are present; and

[0179] (c) washing the treated E. coli from (b) free of the fluoroquinolone antibiotic to result in a fluoroquinolone inactivated S. aureus vaccine.

[0180] Ideally, the time period is from approximately 2-24 hours (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours), preferably 2-4 hours, more preferably approximately 3 hours. The present inventors found that strong T cell responses were observed when the bacteria were treated for 3 hours with the fluoroquinolone antibiotics. They also found that bacteria treated with fluoroquinolone antibiotics for 24 hours were also immunogenic, but the responses were in some cases weaker than those observed for bacteria treated with fluoroquinolone antibiotics for 3 hours. Thus, the treatment time may need adjustment depending on the bacteria being treated and the antibiotic (and associated dosage) being used. In this manner the skilled person can adjust the time to maximise immunogenicity by ensuring the bacterial cells do not lyse, the bacterial antigens remain intact, and there are no live bacteria present.

[0181] Ideally, the bacterial culture has a CFU of approximately lxlO9.

[0182] Ideally, the effective amount of fluoroquinolone antibiotic is from 0.01 to 3mg / ml.

[0183] The B. pertussis isolate in (a) may be frozen B. pertussis stocks. Optionally, the B. pertussis isolate is a B. pertussis Tohama-1 isolate or derivative thereof. Still optionally, the B. pertussis isolate is a clinical isolate, ideally a recent clinical isolate. Other B. pertussis strains may be used.

[0184] Ideally, the fluoroquinolone antibiotic is selected from one or more of ciprofloxacin, gemifloxacin, levofloxacin, moxifloxacin, delafloxacin, norfloxacin and ofloxacin; preferably ciprofloxacin or levofloxacin.

[0185] It will be understood that these methods do not comprise a heat inactivation step or a chemical (aldehyde) inactivation step either before or after the antibiotic or fluoroquinolone treatment step. In this manner, the bacteria are inactivated by treatment with a quinolone antibiotic only. This method involves no pre-treatment or post-treatment with aldehydes, which are known to cross link and denature proteins / antigens, and thereby adversely affect the immunogenicity of the antigens in aldehyde treated vaccine preparations.

[0186] In embodiments of the aspects of the present invention, the present invention extends to improvements in the efficacy of vaccines, for example a Bordetella pertussis or S. aureus vaccine.

[0187] In embodiments of the aspects of the present invention, the vaccine or vaccine composition may further comprise adjuvants, pharmaceutically acceptable carriers, diluents or auxiliaries which may enhance the immunogenicity or effectiveness of the vaccine composition. It will be understood that the vaccine may or may not comprise an adjuvant, such as aluminium salt adjuvant. Further adjuvants are discussed below. In embodiments of the aspects of the present invention, the vaccine or vaccine composition may be provided in accordance with the manner in which it is to be administered. In embodiments of the aspects of the present invention, the vaccine or vaccine composition may be provided in a spray-container, aerosol can or nebuliser for intranasal administration by inhalation. Alternatively, the vaccine or vaccine composition may be provided in a conventional container suitable for administration by injection.

[0188] In embodiments of the aspects of the present invention, the vaccine of the invention, or compositions comprising same, are administered prophylactically to a subject. In certain further embodiments of the aspects of the present invention, the vaccine composition of the present invention, or compositions comprising same, are administered therapeutically. Prophylactic and therapeutic compositions may be administered to subjects in need thereof as required.

[0189] In embodiments of the aspects of the present invention, the subject is a mammal or avian, in particular a human. In embodiments the subject can be an animal.

[0190] In embodiments of the aspects of the present invention, the vaccine or vaccine composition comprises an adjuvant. In embodiments of the aspects of the present invention, adjuvants can be selected from a group of aluminium salts, inulin, algammulin, combination of inulin and aluminium hydroxide, monophosphoryl lipid A (MPL), resiquimoid, muramyl dipeptide (MDP), N-glycolyl dipeptide (GMDP), poly IC, CpG oligonucleotide, resiquimod, aluminium hydroxide with MPL, any water in oil emulsion, any oil in water emulsion that contains one or more of the following constituents: squalene or its analogues, genetic adjuvants (DNA or mRNA expressing vector) or any pharmaceutically acceptable oil, tween-80, sorbitan trioleate, alpha-tocopherol, cholecalciferol or any of the analogues and derivatives of the molecules thereof, or calcium phosphate or any combination of the adjuvants. In embodiments of the aspects of the present invention, the Bordetella pertussis is administered simultaneously with or sequentially to the adjuvant.

[0191] In embodiments of the aspects of the present invention, the vaccine or vaccine composition comprises antibiotic-inactivated bacteria wherein the bacteria has deleted or modified genes coding for various toxins. In embodiments of the aspects of the present invention, the antibiotic-inactivated Bordetella pertussis (AIBP) vaccine of the present invention may involve antibiotic inactivation of B. pertussis strains that have deleted or modified genes coding for one or more of pertussis toxin (PT), dermonecrotic toxin (DNT), tracheal cytotoxin (TCT), adenylate cyclase (ACT), and lipooligosaccharide (LPS) or lipooligosaccharide (LOS).

[0192] Preferred features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless context demands otherwise.

[0193] Brief Description of the Figures

[0194] An embodiment of the present invention will now be described with reference to the following figures which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention wherein:

[0195] Figure 1A-B demonstrate the effect of ciprofloxacin on the growth of B. pertussis in liquid culture. B. pertussis was cultured in S&S medium at a starting concentration of 6xl06(Figure 1A) or 6xl07CFU / ml (Figure IB) with 0-1-0.5 mg / ml of ciprofloxacin or medium only. After 2, 6, 12 and 24 h, live bacteria were quantified by performing CFU counts on BG agar plates.

[0196] Figure 2A-C demonstrate the morphology of ciprofloxacin-inactivated B. pertussis. Representative images ofB. pertussis culture treated with ciprofloxacin at 0.25 mg / ml (Figure 2A), 0.5mg / ml (Figure 2B) or left untreated (Figure 2C). After 24h culture, bacteria were fixed with PFA and stained with anti-B. pertussis-LOS-A..

[0197] Figure 3A-C demonstrate that antibiotic-inactivated B. pertussis (AIBP) enhances expression of MHC class II and co -stimulatory molecules on dendritic cells. Bone marrow-derived dendritic cells (BMDCs) were stimulated for 24 h with AIBP, live B. pertussis 338, heat-killed Bordetella pertussis (HKBP) (bacterium-to-cell ratio of 10:1) or medium. Surface expression MHC class II (MHCII) (Figure 3A), CD80 (Figure 3B) and CD86 (Figure 3C) were evaluated using flow cytometric analysis. Results are expressed as Mean fluorescence intensity (MFI) and show individual values for 4 replicates. Data were analyzed by One-way ANOVA followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01.

[0198] Figure 4A-E demonstrates that AIBP induces production of Thl and Thl7 polarizing cytokines by dendritic cells. BMDCs were stimulated for 24 h with AIBP, live B. pertussis 338, HKBp (bacterium-to-cell ratio of 10:1) or medium and the concentrations of IL-ip (Figure 4A), IL-6 (Figure 4B), IL-23 (Figure 4C), IL-12 (Figure 4D) and TNF (Figure 4E) in supernatants were quantified by ELISA. Data were analyzed by One-way ANOVA followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0199] Figure 5A-C demonstrates immunofluorescence staining of B. pertussis in the lungs after aerosol administration of live B. pertussis or AIBP. Mice were exposed to an aerosol of live B. pertussis (Figure 5A), AIBP (Figure 5B) or PBS (Figure 5C). After 24 h lung section from euthanized mice were stained with anti-B. pertussis LOS-A and goat anti-mouse IgG3- Alexa Fluor 594 antibodies.

[0200] Figure 6A-D demonstrates that aerosol administered AIBP vaccine promotes transient induction of Thl and Thl7 polarizing cytokines in the lungs. Groups of 8 mice were exposed to an aerosol of the AIBP vaccine or live virulent B. pertussis or were immunized i.m. with a wP vaccine or PBS. The concentration of IL-6 (Figure 6A), IL-ip (Figure 6B), IL-23 (Figure 6C) and TNF (Figure 6D) were quantified by ELISA in lung homogenates in groups of 4 mice after 4 and 24 h. Data were analysed by two-way ANOVAs followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0201] Figure 7A-D demonstrates that aerosol administered AIBP vaccine promotes transient induction of IL-6 in the nasal tissue. Mice were immunized as described in Figure 6 and the concentration of cytokines quantified in homogenized lung tissue after 4 and 24 hours. Data were analysed by two-way ANOVAs followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0202] Figure 8A-E demonstrates that the AIBP vaccine does not induce inflammatory cytokines systemically. Mice were immunized as described in Figure 6 and the concentration of cytokines quantified in serum after 4 and 24 hours. Data were analysed by two-way ANOVAs followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0203] Figure 9A-B demonstrates that aerosol immunization of mice with the AIBP vaccine confers protection against B. pertussis infection of the lungs and nasal cavity. Mice were immunized by aerosol administration of AIBP vaccine (once or twice at 0 and 4 weeks), an aP vaccine (twice, 0 and 4 weeks;l / 50 of the human dose) or PBS. Mice were aerosol challenged from a culture containing lxlO9CFU / mL live B. pertussis at week 6. Lung and nasal tissue were collected at day 7, 14 and 21 post challenge bacterial loads quantified by performing CFU counts on lung (Figure 9 A) and nasal tissue (Figure 9B) homogenates.

[0204] Figure 10A-C demonstrates that the AIBP vaccine, but not the aP vaccine, induces respiratory CD4 tissue-resident memory (TRM) cells. Mice were immunized as described in Figure 9. CD4 TRM cells were quantified by flow cytometry on lungs and nasal tissue cells prior to B. pertussis challenge. Flow cytometry analysis showing representative plots of CD69 and CD103 expression on CD45iv_CD44+CD62L- CD4+T cells. Data were analysed by One-way ANOVA followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0205] Figure 11A-C demonstrates that the AIBP vaccine, but not the aP vaccine, induces respiratory CD4 TRM cells 7 days after live B. pertussis challenge. Mice were immunized as described in Figure 9. CD4 TRM cells were quantified by flow cytometry in the lungs and nasal tissue 7 days after challenge with live B. pertussis as described and gated in Figure 10. Data were analysed by One-way ANOVA followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0206] Figure 12A-E demonstrates that the AIBP vaccine, but not the aP vaccine, induces B. pertussis -specific IL-17- and IFN-y-producing CD4 cells in nasal tissues and lung of immunized mice. Mice were immunized as described in Figure 9. Lung or nasal tissue cells were stimulated for 16 hours with HKBp (105CFU / mL), anti-CD28 and anti-CD49d (lpg / mL). IL-17- and IFN-y-producing CD4 cell responses in the respiratory tract of immunized mice with AIBP or aP prior to challenge with live B. pertussis were analysed using flow cytometry. Data were analyzed by One-way ANOVA followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0207] Figure 13A-E demonstrates that the AIBP vaccine-induces B. pertussis -specific IL-17- and IFN-y-producing CD4 cells are enhanced in the respiratory tract after B. pertussis challenge. Mice were immunized as described in Figure 9. IL-17- and IFN-y-producing CD4 cell responses in the respiratory tract of immunized mice with AIBP or aP vaccine 7 days after challenge with live B. pertussis were analysed using flow cytometry. Data were analysed by One-way ANOVA followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0208] Figure 14A-C demonstrates that aerosol immunization of mice twice with the AIBP vaccine generate B. pertussis-specific IgGl, IgG2c in serum and IgA in nasal associated lymphoid tissue (NALT). Mice were immunized as described in Figure 9. Figure 14A demonstrates that B. pertussis-specific IgGl and Figure 14B shows IgG2c were quantified in serum by ELISA. Figure 14C demonstrates that NALT cells were cultured for 72 h with sonicated B. pertussis (sBP; 5pg / ml), the supernatants were collected and B. pertussis-specific IgA quantified by ELISA.

[0209] Figure 15A-B demonstrates that the AIBP vaccine confers a higher level of protection against nasal infection than a wP vaccine or previous infection. Mice were immunized by aerosol administration of AIBP vaccine (twice at 0 and 4 weeks), wP vaccine (twice, 0 and 4 weeks;l / 50 of the human dose) or PBS. The protection level of immunized mice were compared with convalescent mice; convalescent mice were defined as mice that were >60 days post B. pertussis challenge on the day of the second dose of vaccine for immunized mice. Mice (immunized and convalescent) were aerosol challenged with lxlO9CFU / mL live B. pertussis at week 6. Lung and nasal tissue were collected at day 7, 14 and 21 post challenge bacterial loads quantified by performing CFU counts on lung (Figure 15A) and nasal tissue (Figure 15B) homogenates.

[0210] Figure 16A-C demonstrates that the AIBP vaccine induces IgA, but lower concentrations of IgGl and IgG2c than the wP vaccine or previous infection. Mice were immunized as described in Figure 15. On the day prior to B. pertussis challenge, concentrations of B. pertussis-specific IgA in NALT supernatants (Figure 16A) and B. pertussis-specific IgGl (Figure 16B) and IgG2c (Figure 16C) in serum were quantified by ELISA as described in Figure 14.

[0211] Figure 17A-B demonstrates that the AIBP vaccine induces B. pertussis-specific Thl and Thl7 responses in lymph node (LN), which are stronger than those induced with the wP vaccine.

[0212] On the day prior to B. pertussis challenge single-cell suspensions from cervical and inguinal LN and spleen cells (from naive mice as a source of additional antigen presenting cells) at a ratio of 9:1 were stimulated with HKBp, or medium at a concentration of 2 x 106 / mL. After 3 days of culture the concentrations of IL-17 (Figure 17A) and IFN-y (Figure 17B) in supernatants were quantified by ELISA. Data were analyzed by two-way ANOVAs followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0213] Figure 18A-E demonstrates that the AIBP vaccine induces B. pertussis-specific IL-17 and IFN-y-secreting CD4 cells in the lungs and nasal tissue, which are stronger than those induced with the wP vaccine. Mice were immunized as described in Figure 15. On the day prior to B. pertussis challenge, T cell responses in lung (A) and nasal tissue (B) were analysed by flow cytometry as described in Figure 10. Lung or nasal tissue cells were stimulated as describe in Figure 12 and IL-17- and IFN-y-producing CD4 cell were analyzed by intracellular cytokine staining and flow cytometry. Data were analyzed by One-way ANOVA followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0214] Figure 19A-B demonstrates that prior immunization of mice with aP vaccines does not affect the efficacy of the AIBP vaccine against lung and nasal infection. Mice were immunized i.m. with aP vaccine (twice at 0 and 4 weeks; 1 / 50 of the human dose) followed by administration of AIBP vaccine (by aerosol once at 8 weeks or twice at 8 and 12 weeks) or given the AIBP (once or twice) or PBS. Mice were aerosol challenged with live B. pertussis at week 14. Lung and nasal tissue were collected at day 7, 14 and 21 post challenge bacterial loads were quantified by performing CFU counts on lung (Figure 19A) and nasal tissue (Figure 19B) homogenates.

[0215] Figure 20A-C demonstrates that the AIBP vaccine generates B. pertussis-specific IgA in NALT and IgG2c in the serum and this is not compromised by prior immunization with the aP vaccine. Mice were immunized as described in Figure 19. On the day prior to B. pertussis challenge, concentrations of B. pertussis-specific IgA in NALT supernatants (Figure 20A) and B. pertussis-specific IgGl (Figure 20B) and IgG2c (Figure 20C) in serum were quantified by ELISA as described in Figure 14.

[0216] Figure 21A-B demonstrates that the AIBP vaccine, but not the aP vaccine, induces B. pertussis -specific T cell responses in LN and this is not affected by prior immunization with the aP vaccine. Mice were immunized as described in Figure 19. Concentrations of IL- 17 and IFN-y were quantified by ELISA as described in Figure 17. Data were analyzed by two- way ANOVAs followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0217] Figure 22A-B demonstrates that the AIBP vaccine, but not the aP vaccine, induces respiratory CD4 TRM cells and this is not affected by prior immunization with the aP vaccine. Mice were immunized as described in Figure 19. On the day prior to B. pertussis challenge, T cell responses in the respiratory tract were analysed by flow cytometry as described in Figure 10. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 by one-way ANOVA followed by Tukey’s test for multiple comparisons.

[0218] Figure 23A-D demonstrates that the AIBP vaccine, but not the aP vaccine, induces IL- 17 and IFN-y-secreting respiratory CD4 cells and this is not affected by prior immunization with the aP vaccine. Mice were immunized as described in Figure 19. On the day prior to B. pertussis challenge, lung or nasal tissue cells were stimulated for 16 hours with HKBp (105CFU / mL), anti-CD28 and anti-CD49d (lpg / mL) and IL-17- and IFN-y- producing CD4 cell were analyzed by intracellular cytokine staining and flow cytometry. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 by One-way ANOVA followed by Tukey’s test for multiple comparisons.

[0219] Figure 24A-B demonstrates that intranasal administration of AIBP vaccine confers protection against infection with B. pertussis of the lung and nose. Mice were immunized i.n. with low, medium and high doses (3xl05, 3xl06or 3xl07CFU) of AIBP vaccine (twice at 0 and 4 weeks) or PBS or were infected intranasally with live B. pertussis (convalescent). Mice (immunized and convalescent) were challenged at week 6 by exposure to an aerosol from a culture containing lxlO9CFU / mL live B. pertussis. Lung and nasal tissue were collected at intervals after challenge and bacterial loads quantified by performing CFU counts on lung (Figure 24A) and nasal tissue (Figure 24B) homogenates.

[0220] Figure 25A-C demonstrates that intranasal administration of the AIBP vaccine generates B. pertussis-specific IgA in NALT, IgGl and IgG2c in the serum. Mice were immunized as described in Figure 24. On the day prior to B. pertussis challenge, concentrations of B. pertussis-specific IgA in NALT supernatants (Figure 25A) and B. pertussis-specific IgGl (Figure 25B) and IgG2c (Figure 25C) in serum were quantified by ELISA as described in Figure 14.

[0221] Figure 26A-B demonstrates that intranasal administrated the AIBP vaccine induces respiratory CD4 TRM cells in the nose. Mice were immunized as described in Figure 24. On the day prior to B. pertussis challenge, T cell responses in nasal tissue (Figure 26A) and lungs (Figure 26B) were analysed by flow cytometry as described in Figure 10. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 by one-way ANOVA followed by Tukey’s test for multiple comparisons.

[0222] Figure 27A-D demonstrates that intranasal administration of the AIBP vaccine induces IL-17 and IFN-y-secreting respiratory CD4 cells. Mice were immunized as described in Figure 24. On the day prior to B. pertussis challenge, lung or nasal tissue cells were stimulated as described in Figure 23 and IL-17- and IFN-y-producing CD4 cell were analysed by intracellular cytokine staining and flow cytometry. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 by One-way ANOVA followed by Tukey’s test for multiple comparisons.

[0223] Figure 28. Effect of levofloxacin and chloramphenicol on the growth of B. pertussis in liquid culture. B. pertussis was cultured in Stainer-Scholte (S&S) medium at a starting concentration of 6xl07CFU / ml with 0.25-0.75 mg / ml (A), 1-3 mg / ml (B) Levofloxacin or 25- lOOpg of chloramphenicol (C) and medium only. After 3, 6, 12 and 24 h, live bacteria were quantified by performing CFU counts on Bordet-Gengou (BG) agar plates.

[0224] Figure 29. Effect of ciprofloxacin and levofloxacin on the growth of Staphylococcus aureus in liquid culture. S. aureus was cultured in nutrient broth medium at a starting concentration of 3xlO10CFU / ml with 0.1-0.5 mg / ml (A) or 1-3 mg / ml (B) of ciprofloxacin or 0.1-0.5 mg / ml (C) or 1-3 mg / ml (D) of levofloxacin or medium only. Live bacteria were quantified by performing CFU counts on Nutrient agar plates at the indicated timepoints of culture.

[0225] Figure 30: Morphology of live and antibiotic inactivated B. pertussis. B. pertussis 338 was treated with A) 1 mg / ml levofloxacin or B) 100 pg / mL chloramphenicol for 24 h. C) heat- killed B. pertussis (HKBpj. D) live untreated B. pertussis. Figure 31: Morphology of live and antibiotic-inactivated E. coli. E. coli Top 10 was treated with A) 1 mg / ml levofloxacin, B) 0.25mg / ml ciprofloxacin, C) 100 pg / mL kanamycin or D) 100 pg / mL streptomycin for 24h. E) Live untreated E.coli.

[0226] Figure 32: Morphology of live and antibiotic-inactivated Staphylococcus aureus. S. aureus was treated with A) 1 mg / ml levofloxacin, B) 1 mg / ml ciprofloxacin, C) 100 pg / mL penicillin, and D) 100 pg / mL streptomycin for 24 h. E) Live untreated S. aureus.

[0227] Figure 33. B. pertussis inactivated with ciprofloxacin and levofloxacin (fluoroquinolone family antibiotics), but not chloramphenicol, induces B. pertussisspecific IL-17- and IFN-y-producing CD4 tissue-resident memory T (TRM) cells in the lungs of aerosol immunized mice. B. pertussis 338 was inactivated with 0.25 mg / ml ciprofloxacin for 3 h (Bp-Cip-3h) or 24 h (Bp-Cip-24h), 1 mg / mL levofloxacin for 3h (Bp-Lev- 3h) or 100 pg / mL chloramphenicol for 3h (Bp-Chl-3h). C57BL / 6 mice were immunized once by aerosol administration of each of antibiotic inactivated B. pertussis preparations or with PBS. Immune cells were prepared from lungs 14 days post-vaccination and stained with antibodies specific for surface markers, CD3, CD4, CD8, CD44, CD62L, CD69 and CD103 for quantification of total CD4 T cells (A) or tissue resident memory CD4 T (TRM) cells (B) by flow cytometric analysis. Lung cells were stimulated for 16 hours with heat-killed B. pertussis (HKBp; 105CFU / mL), anti-CD28 and anti-CD49d (lpg / mL). IL-17 (C) and IFN-y (D) producing CD4 TRM cell responses in the lungs of immunized mice were analysed using flow cytometry. Data were analysed by Two-way ANOVA followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0228] Figure 34. B. pertussis inactivated with ciprofloxacin and levofloxacin (fluoroquinolone family antibiotics), but not chloramphenicol, induces CD4 TRM cells in the nasal tissue of aerosol immunized mice. B. pertussis 338 was inactivated with 0.25 mg / ml ciprofloxacin for 3 h (Bp-Cip-3h) or 24 h (Bp-Cip-24h), 1 mg levofloxacin for 3h (Bp- Lev-3h) or 100 pg / mL chloramphenicol for 3h (Bp-Chl-3h). C57BL / 6 mice were immunized once by aerosol administration of each of antibiotic inactivated B. pertussis preparations or with PBS. Immune cells were prepared from nasal tissue 14 days post-vaccination and stained with antibodies specific for surface markers, CD3, CD4, CD8, CD44, CD62L, CD69 and CD103 for quantification of total CD4 T cells (A) or tissue resident memory CD4 T (TRM) cells (B) by flow cytometric analysis. Data were analysed by Two-way ANOVA followed by Tukey’s test for multiple comparisons. **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0229] Figure 35. B. pertussis inactivated with ciprofloxacin and levofloxacin (fluoroquinolone family antibiotics), but not chloramphenicol, induces B. pertussisspecific IL-17- and IFN-y-production by spleen cells of aerosol immunized mice. B. pertussis 338 was inactivated with 0.25 mg / ml ciprofloxacin for 3 h (Bp-Cip-3h) or 24 h (Bp- Cip-24h), 1 mg levofloxacin for 3h (Bp-Lev-3h) or 100 pg / mL chloramphenicol for 3h (Bp- Chl-3h). C57BL / 6 mice were immunized once by aerosol administration of each of antibiotic inactivated B. pertussis preparations or with PBS. On day 14 post-vaccination, spleen cells were prepared from immunized mice were stimulated for 72 h at lxlO6cells / well, with HKBp (lxlO5CFUs / ml) or medium as a negative control. Concentrations of IFN-y (A) and IL-17 (B) in supernatants were quantified by ELISA. Data were analyzed by Two-way ANOVA followed by Tukey’s test for multiple comparisons. ****p< 0.0001.

[0230] Figure 36: Aerosol immunization of mice with Staphylococcus aureus inactivated with levofloxacin induces respiratory CD4 TRM cells in lung and nasal tissue. .S', aureus was treated with 1 mg / ml levofloxacin for 3 h. C57BL / 6 mice were immunized once by aerosol administration of levofloxacin inactivated S. aureus (Sa-Lev) or with PBS. Immune cells were prepared from nasal tissue (A-B) and lungs (C-D) 14 days post-vaccination and stained with antibodies specific for surface markers, CD3, CD4, CD8, CD44, CD62L, CD69 and CD103 for quantification of total CD4 T cells (A, C) or tissue resident memory CD4 T (TRM) cells (B, D) by flow cytometric analysis. Lung cells were stimulated for 16 hours with heat-killed S. aureus (HKSa; 105CFU / mL), anti-CD28 and anti-CD49d (1 pg / mL). IL-17 (E) and IFN-y (F) producing CD4 TRM cell responses in the lungs of immunized mice were analysed using flow cytometry. Data were analysed by Unpaired ttest. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0231] Figure 37: Aerosol immunization of mice with Staphylococcus aureus inactivated with levofloxacin induces S. aureus-specific IFN-y and IL-17 in the spleen. .S', aureus was treated with 1 mg / ml levofloxacin for 3 h. C57BL / 6 mice were immunized once by aerosol administration of levofloxacin-inactivated S. aureus (Sa-Lev) or with PBS. On day 14 postvaccination, spleen cells prepared from immunized mice were stimulated for 72 h at lxlO6cells / well, with heat-killed S. aureus (HKSa) (lxlO5CFUs / ml) or medium as a negative control. Concentrations of IFN-y (A) and IL-17 (B) in supernatants were quantified by ELISA. Data were analyzed by Two-way AN OVA followed by Tukey’s test for multiple comparisons. **p < 0.01, ****p< 0.0001.

[0232] Figure. 38. The AIBP vaccine does not replicate or produce pertussis toxin in vivo. Mice were exposed to an aerosol of live B. pertussis or AIBP vaccine. A) CFU were assessed in lungs and nasal tissue after 0, 24 and 72 hours, B) The concentration of pertussis toxin (PT) was quantified in lung by ELISA.

[0233] Figure 39. The AIBP vaccine is more effective than the whole cell pertussis (wP) vaccine in promoting dendritic cell (DC) maturation and production of Thl and Thl7 polarizing cytokines. A,B, Bone marrow-derived dendritic cells (DCs) were stimulated for 24 hours with AIBP or wP vaccine (bacterium-to-cell ratio of 2:1) or medium. A) Surface expression MHC class II (MHCII), CD40 and CD80 was evaluated using flow cytometric analysis. Results are expressed as Mean fluorescence intensity (MFI) and show individual values for 4 replicates. B) Concentrations of IL-lp, IL-12p70 and IL-23 in supernatants were quantified by ELISA. C) Enriched CD4 T cells (with residual antigen presenting cells) from spleens of convalescent mice were cultured (2.5 x 106 / ml) with AIBP or the wP vaccine (at concentrations equivalent to 1 xlO7bacteria / ml). After 3 days of culture, the concentrations of IL-17 in supernatants were quantified by ELISA. Data were analyzed by One-way ANOVA followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0234] Figure 40. AIBP vaccine-activated APC stimulate B. pertussis -specific Thl and Thl7 cells in vitro. CD4 T cells purified from the spleen of B. pertussis convalescent mice were cultured with APC (irradiated spleen cells, 2 xl06 / ml) and increasing concentrations of AIBP or HKBp. After 3 days, IL-17 and IFN-y production was quantified in supernatants by ELISA. Data were analyzed by two-way ANOVAs followed by Tukey’s test for multiple comparisons. **p < 0.001, ****p< 0.0001.

[0235] Figure 41. IL-17-secreting CD4 T cells mediate protection induced with AIBP vaccine. Mice were immunized once by aerosol administration of AIBP vaccine and treated with anti- CD4 (aCD4), anti-IL-17 (aIL-17) or an isotype (Iso) control antibody the day before and every 3 days after aerosol challenge with live B. pertussis at week 6. A) CFU counts were performed on lung and nasal tissue at day 7, 14 and 21 post challenge. B) On the day of but prior to challenge with live B. pertussis, CD4 TRM cells (CD45iv_CD4+CD44+CD62L“CD69+CD103+ / ) were quantified in lungs and nasal tissue by flow cytometric analysis. C) On the day of but prior to B. pertussis challenge, Siglec-F+neutrophils were quantified in lung and nasal tissue cells by flow cytometry. Data were analyzed by two-way ANOVAs followed by Tukey’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

[0236] Figure 42. Intranasal administration with different doses of the AIBP vaccine confers protection against infection with B. pertussis of the lung and nose. Mice were immunized i.n. with low, medium and high doses (3xl05, 3xl06or 3xl07CFU) of AIBP vaccine (twice at 0 and 4 weeks) or PBS or were infected intranasally with live B. pertussis (convalescent). Mice (immunized and convalescent) were challenged at week 6 by exposure to an aerosol from a culture containing lxlO9CFU / mL live B. pertussis. Lung and nasal tissue were collected at intervals after challenge and bacterial loads quantified by performing CFU counts on lung (A) and nasal tissue (B) homogenates.

[0237] Detailed Description of the Invention

[0238] The present inventors describe a new platform approach to vaccination against bacterial infections, involving, antibiotic inactivated bacteria or bacteria inactivated by an antibacterial compound, for example, involving respiratory delivery of antibiotic inactivated bacteria or bacteria inactivated by an antibacterial compound. The inventors have demonstrated that a bacteria inactivated with fluoroquinolone antibiotics or an antibacterial compound can be effectively used in a vaccine or vaccine composition against infection from the bacteria or in the treatment and / or prophylaxis of a disease associated with the bacteria. In particular, this new platform vaccination approach of the present invention provides effective vaccines and vaccine compositions for bacteria that infect mucosal sites and particularly that result in respiratory tract infections and / or diseases of the respiratory tract. The inventors provide proof-of-principle with ciprofloxacin treated B. pertussis, which they demonstrate to be a safe and effective approach for conferring protection against B. pertussis infection in a mouse model. Further experimental data has extended this platform approach to other gram-positive and gram-negative bacteria, including S. aureus and E. coli and to other fluoroquinolone antibiotics. Delivery of antibiotic-inactivated B. pertussis (AIBP) to mice by aerosol route promoted the induction of secretory IgA and TRM cells in the lung and nasal tissue and conferred sterilizing immunity against B. pertussis infection of the lungs and nasal tract. A single immunization of mice with an aerosol of AIBP vaccine substantially reduced the bacterial load in the nose after live B. pertussis challenge, and two immunizations conferred complete protection against lung and nasal infection. Furthermore, the AIBP vaccine induced T cells that remained in the lung and nasal tissue as resident memory T (TRM) cells. The AIBP vaccine also induced potent B. pertussis-specific antibody response in the circulation and in the respiratory tract. In contrast, two doses of a parenterally delivered aP vaccine induced B. pertussis-specific antibodies in the blood, but not in the respiratory tract, and also failed to generate respiratory TRM cells or to prevent nasal infection with B. pertussis. Collectively the present data provide evidence of a novel mucosally-delivered vaccine approach that is safe and highly effective for inducing sterilizing immunity against mucosal pathogens in humans. Furthermore, the AIBP is considerably more effective than the current aP vaccine and safer than the older wP vaccine.

[0239] It is generally accepted that the current aP vaccines are suboptimal, failing to prevent infection of the nasal tract and allowing transmission of B. pertussis from immunized individuals [7, 8], While the aP vaccine induce potent circulating IgG and Th2 responses, because they are administered parenterally with alum as the adjuvant, they fail to induce IgA, Thl / Thl7 cells or TRM cells in the respiratory tract [7], In addition, antibody responses wane quickly after immunization

[0029] , and immunity can be evaded through the emergence of antigenically divergent circulating B. pertussis strains because of vaccine-driven antigen variation in B. pertussis

[0030] , Therefore, there is a need to develop a vaccine that induces sterilizing immunity by inducing local T cells and antibodies (IgG and IgA) in the respiratory tract, but also better immunological memory, including memory T and B cells in the lungs and nasal tissue. The current acellular pertussis (aP) vaccine is suboptimal because it fails to induce Thl or Thl7 responses or tissue-resident memory (TRM) CD4 T cells and does not prevent nasal colonization with B. pertussis. The aerosol-delivered antibiotic-inactivated whole bacteria vaccine of the present invention promotes the induction of Thl, Thl7 and TRM cells and protects against lung and nasal infection. It also addressed the broader problem of low immunogenicity of non-replicating injected vaccines in general. Attempts to develop new vaccines against pertussis have focused on live attenuated vaccines, outer membrane vesicles (OMV) vaccines or subunit vaccines administered with novel adjuvants. The attenuated vaccines are the most developed, with BPZE1 now in phase 2 clinical trials, having been shown to be safe in adult human volunteers in phase I trials

[0031] , Studies in mice have shown that the attenuated vaccine can induce mucosal IgA and respiratory TRM cells and prevent nasal and lung infection of mice with B. pertussis

[0032] , However, the limitations of attenuated vaccines include a failure to "take” in a proportion of vaccines because of pre-existing antibodies to B. pertussis from previous vaccination or possibly previous infection

[0033] , Although three of the major toxins, PT, dermonecrotic toxin and tracheal toxin have been genetically modified or removed from BPZE1, the bacteria are live and could transiently colonize the nasopharynx with the potential persist in immunized individual, especially the immunocompromised. Live attenuated vaccines can change the composition of the nasal microbiota, which can potentially modulate immune responses in the nose early in life. An intranasal live attenuated influenza vaccine has been shown to alter the microbial community structure, diversity and composition, including increases in the relative abundances of Staphylococcus and Bacteroides genera

[0034] ,

[0240] The outer membrane vesicles [OMV] vaccines have shown some promising results in mouse models and are capable of preventing lung and nasal infection with B. pertussis [35, 36], However, they do not offer strong sterilising immunity to B. pertussis. Finally, the aP vaccine with novel adjuvants, including the TLR9 agonist CpG, the TLR2 and STING agonists LP-GMP are capable of inducing Thl and Thl7 responses as well as antibodies and confer protection against nasal and lung infection with B. pertussis

[0024] . The limitation of the subunit approach includes possible reduced efficacy against circulating mutant strains of B. pertussis with deletion of or mutation in protective antigens in the vaccine.

[0241] The inventors’ approach of using ciprofloxacin-treated B. pertussis has significant advantages over current aP vaccines and combines the benefits of live attenuated pertussis vaccines and wP vaccines, but with reduced risks and increased immunogenicity, especially when delivered mucosally. Ciprofloxacin is widely used as a broad-spectrum antibiotic that permeates bacterial membrane and kills the bacteria by arresting cell division by inhibiting the function of DNA gyrase and / or DNA Topoisomerase IV

[0037] , The inventors’ results demonstrate that ciprofloxacin requires less than 2-4 hours to completely inactivate B. pertussis in vitro. The cipro floxacin-inactivated B. pertussis could be detected in the lung 24 h after aerosol administration, but as cell division is inhibited, the bacteria do not colonize the lungs or nasal cavity and does not induce cytokines systemically

[0242] The inventors demonstrate that B. pertussis enlarge following ciprofloxacin treatment, and this is likely to facilitate uptake of the killed bacteria by antigen presenting cells. Furthermore, the AIBP vaccine induces production of the T cell polarizing IL-1 p, IL-6, IL-12 and IL-23 by DCs and locally at the site of immunization in the respiratory tract, resulting in highly effective priming of Thl and Thl7 cells, which play a key role in protective immunity to B. pertussis. The inventors demonstrate that the AIBP vaccine delivered to mice by the aerosol route are very effective at inducing respiratory T cell responses, including IL-17 and IFN-y-secreting respiratory CD4 TRM cells. Although two doses were required to induced potent serum IgG and mucosal IgA, a single dose of the AIBP vaccine induced respiratory TRM cells and conferred high level of protection against B. pertussis infection of the lung and nasal tract. Although the inventors do not overrule a role for antibodies, their findings suggest that T cells are major mediators of protective immunity induced with the AIBP vaccine. Since TRM cells persists in respiratory tissue, this suggests that immunological memory and protective immunity induced with the AIBP vaccine will be sustained, giving it a further advantage over the current aP vaccines.

[0243] One of the possible risks of whole bacterial vaccines, whether they be attenuated or inactivated, is reactogenicity from systemic inflammatory responses. The wP vaccine are associated with adverse events, including fevers and febrile seizure, and this led to their demise in most high- and middle-income countries. Here the inventors show that parenteral immunization of mice with the wP vaccine resulted in highly significant increase in the concentration of proinflammatory cytokines IL-ip, IL-6 and TNF in the circulation. In contrast, the mucosally-delivered AIBP vaccine, although inducing beneficial respiratory innate immune responses that prime protective T cells, this did not result in enhanced circulating cytokines, suggesting that the vaccine has a good safety profile. Since LPS is one of the major mediators of inflammation and vaccine reactogenicity, it should be possible to further reduce any risk associated with the AIBP vaccine by preparing it from B. pertussis with reduced LOS content

[0038] and by deleting or mutating other B. pertussis toxins.

[0244] The existing aP vaccine does not induce B. pertussis-specific Thl or Thl7 cells or respiratory TRM cells to prevent infection of nasal tract with B. pertussis and there is some evidence that it may suppress induction of CD4 TRM cells and compromise bacterial clearance post live B. pertussis challenge. The aerosol-delivered AIBP vaccine of the present invention has significant advantages over current parenterally delivered aP vaccines. Most importantly it protects against nasal as well as lung infection and should therefore prevent community transmission of B. pertussis. Existing pertussis vaccines fail to prevent nasal colonization and transmission with B. pertussis, supporting the evidence of asymptomatic transmission of B. pertussis from fully vaccinated individuals. Importantly and surprisingly, the vaccine of the present invention prevents or reduces transmission of B. pertussis from immunized individuals to others. As well as inducing IgG antibodies, it generates Thl and Thl7 cells, and because it is delivered to the respiratory tract is capable of inducing slgA and T RM cells, which is crucial for long term local immunity to B. pertussis in the upper and lower respiratory tract. Furthermore, the immunogenicity and protective efficacy of AIBP vaccine was not affected by previous immunization with two doses of the aP vaccine. It has been demonstrated that the aP vaccine primes Th2 responses and there have been suggestions that the aP vaccine may set the immune response to a type 2 phenotype, making it more difficult to shift to the more protective Thl / Thl7 cells and TRM cells. The data provided by the present inventors shows that the AIBP vaccine is highly effective at inducing Thl / Th 17 and TRM cells, more effective than an injected wP vaccine, and not affected by priming with the Th2 -inducing aP vaccine.

[0245] Another significant advantage of the vaccine platform of the present invention is the ease and cost of production, making it especially attractive for developing countries. Furthermore, respiratory administration of the vaccine delivery with a simple nebulizer or as a nasal spray would allow needle-free and pain-free delivery, improving compliance and vaccine uptake. Because of the multiple antigens expressed by the AIBP vaccine, it has the capacity to protect against circulating strains with mutations or deletions in B. pertussis antigens present in the aP vaccine. It also has the versatility to be adapted to cope with strains of B. pertussis that may emerge in the future. Therefore, this new technology has considerable potential for the development of a more effective vaccine against infection with B. pertussis and other gramnegative and gram-positive bacteria in human and animals.

[0246] The present invention further extends to vaccines and vaccine compositions comprising antibiotic inactivated bacteria or bacteria inactivated by an antibacterial compound wherein the bacteria has deleted or modified genes coding for various toxins. The antibiotic inactivated Bordetella pertussis (AIBP) vaccine of the present invention may involve antibiotic inactivation of B. pertussis strains that have deleted or modified genes coding for one or more of pertussis toxin (PT), dermonecrotic toxin (DNT), tracheal cytotoxin (TCT), adenylate cyclase (ACT), and lipooligosaccharide (LPS) or lipooligosaccharide (LOS). Pertussis toxin (PT) is a major virulence factor of B. pertussis that is responsible for many of the severe symptoms of whooping cough. Dermonecrotic toxin (DNT) is a toxin produced by B. pertussis that can contribute to tissue damage. Tracheal cytotoxin (TCT) is a toxin produced by B. pertussis that can damage the trachea. Adenylate cyclase (ACT) is a toxin is a toxin produced by B. pertussis that modulates host immune responses. Lipooligosaccharide (LPS) or lipooligosaccharide (LOS) from B. pertussis has endotoxic activity and promotes inflammatory responses.

[0247] Definitions

[0248] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.

[0249] Throughout the specification, unless the context demands otherwise, the terms 'comprise’ or 'include’, or variations such as 'comprises’ or 'comprising’, 'includes’ or 'including’ will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0250] As used herein, terms such as "a", "an" and "the" include singular and plural referents unless the context clearly demands otherwise. Thus, for example, reference to "an active agent" or "a pharmacologically active agent" includes a single active agent as well as two or more different active agents in combination, while references to "a carrier" includes mixtures of two or more carriers as well as a single carrier, and the like.

[0251] Throughout this description, the antibiotic will be recited as a fluoroquinolone antibiotic, but it will be understood that a quinolone antibiotic may also be used, and, in any context, these terms are interchangeable. Furthermore, any antibiotic, not limited to a quinolone or fluroquinolone antibiotics, which target metabolic pathways, protein synthesis, essential bacterial enzymes involved in DNA replication and repair, permeates bacterial membrane and / or kills the bacteria by arresting cell division by inhibiting the function of DNA gyrase and / or DNA Topoisomerase IV may be used in the present invention. The bacteria inactivated by the fluoroquinolone antibiotic is not lysed, i.e. bacterial lysis does not occur, and retains an intact morphology compared to untreated bacteria. Furthermore, the bacterial antigen structure remains intact, such that at least one or more bacterial antigens are not modified.

[0252] Throughout this description, the terms 'vaccine’, 'vaccine composition’ and 'immunogenic composition’ are used and defined below and it will be understood that these terms are interchangeable.

[0253] Vaccine Composition / Vaccine

[0254] As used herein, the term "vaccine composition” means any composition containing an immunogenic determinant which stimulates the immune system in a manner such that it can better respond to subsequent challenges or pathogenic infections.

[0255] It will be appreciated that a vaccine usually contains an immunogenic determinant and optionally an adjuvant, the adjuvant serving to non-specifically enhance the immune response to the immunogenic determinant.

[0256] In the present invention, the antigen may be derived from a bacteria such as B. pertussis, S. aureus or E.coli.

[0257] Immunogenic composition

[0258] By immunogenic composition is meant a composition capable of inducing an immune response when introduced to a subject.

[0259] Administration of vaccine compositions

[0260] The active ingredients used in the present invention, in particular vaccine components and even more particularly antibiotic-inactivated B. pertussis (AIBPj vaccine components, as described herein can be administered separately to the same subject, optionally sequentially, or can be co-administered simultaneously as a pharmaceutical, immunogenic or vaccine composition. The pharmaceutical composition will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected depending on the intended route of administration. In certain embodiments, the vaccine compositions of the present invention may comprise an adjuvant. In certain embodiments, the adjuvant is selected from the group consisting of, but not limited to Quil A, Detox, ISCOMs, genetic adjuvants (DNA or RNA expressing vector) and squalene. Further suitable adjuvants include mineral gels or an aluminium salt such as aluminium hydroxide or aluminium phosphate, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, or may be cationically or anionically derivatised saccharides, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A (MPL), lipid A derivatives (e.g. of reduced toxicity), 3- O-deacylated MPL, quil A, Saponin, QS21, Freund's Incomplete Adjuvant (Difco Laboratories, Detroit, MI), Merck Adjuvant 65 (Merck and Company, Inc., USA), AS-2, AS01, AS03, AS04, AS15 (GSK, USA), MF59 (Novartis, Sienna, Italy), CpG oligonucleotides, bioadhesives and mucoadhesives, microparticles, liposomes, outer membrane vesicles, polyoxyethylene ether formulations, polyoxyethylene ester formulations, muramyl peptides or imidazoquinolone compounds. In embodiments, adjuvants can be selected from a group of aluminium salts, inulin, algammulin, combination of inulin and aluminium hydroxide, monophosphoryl lipid A (MPL), resiquimoid, muramyl dipeptide (MDP), N-glycolyl dipeptide (GMDP), poly IC, CpG oligonucleotide, resiquimod, aluminium hydroxide with MPL, any water in oil emulsion, any oil in water emulsion that contains one or more of the following constituents: squalene or its analogues or any pharmaceutically acceptable oil, tween-80, sorbitan trioleate, alphatocopherol, cholecalciferol or any of the analogues and derivatives of the molecules thereof, or calcium phosphate or any combination of the adjuvants.

[0261] The vaccine of the present invention may be administered to a patient in need of treatment via any suitable route. Typically, a vaccine or vaccine composition of the present invention can be administered parenterally by injection or infusion. Examples of preferred routes for parenteral administration include, but are not limited to; intravenous (IV), intracardial, intraarterial, intraperitoneal (IP), intramuscular (IM), intracavity, subcutaneous (SC / SQ), transmucosal, inhalation, aerosol or transdermal / intradermal (ID). Typically, parenteral administration is by injection or infusion.

[0262] Routes of administration may further include topical and enteral (oral), targeting gastrointestinal or urinary / reproductive tract mucosal surfaces, for example, mucosal (including pulmonary), oral, nasal, rectal, vaginal. A preferred embodiment involves oral delivery, by inhalation or via nasal mucosa, preferably aerosol delivery of the vaccine of the present invention. Aerosol / intranasal delivery is discussed in the section below.

[0263] Where the composition is delivered as an injectable composition, for example in intravenous, intradermal or subcutaneous application, the active ingredient can be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer’s injection or, Lactated Ringer’s injection. Preservatives, stabilisers, buffers, antioxidants and / or other additives may be included, as required.

[0264] The composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood.

[0265] Examples of the techniques and protocols mentioned above and other techniques and protocols which may be used in accordance with the invention can be found in Remington’s Pharmaceutical Sciences, 18th edition, Gennaro, A.R., Lippincott Williams & Wilkins; 20th edition ISBN 0-912734-04-3 and Pharmaceutical Dosage Forms and Drug Delivery Systems; Ansel, H.C. et al. 7th Edition ISBN 0-683305-72-7.

[0266] Aerosol Delivery

[0267] A preferred embodiment involves aerosol delivery of the vaccine of the present invention. The term "aerosol” as used herein is meant to refer to dispersions in air of solid or liquid particles. In general, such particles have low settling velocities and relative airborne stability. In certain embodiments, the particle size distribution is between 0.01 um and 15 um. Aerosol delivery devices include the basic pressurized metered-dose inhaler and nebulizer as well as numerous types of inhalers and devices, including valved holder chambers, dry powder inhalers, soft mist inhalers, nasal sprays, as well as smart inhalers and nebulizers. The term "nebulizer” as used herein is meant to refer to any device that disperses an agent as an aerosol. In certain examples, the device generates an aerosol comprising particles that are between about 0.01-15 microns in size. In preferred examples, when a vaccine formulation or immunogenic composition is applied to the device, the resulting aerosol contains the vaccine and can deliver it into the nasal tract and / or lungs of a subject by normal breathing. Mucosal Delivery

[0268] Traditional routes of mucosal immunization include oral and nasal routes. Other routes for inducing mucosal immunity include the rectal, vaginal, nasal, pulmonary, oral, buccal, sublingual, perlingual and transcutaneous routes. In embodiments, the vaccine composition is administered via intranasal, pulmonary, oral, sublingual, transcutaneous, rectal or vaginal mucosal routes. In embodiments, the preferred route of administration is intranasal.

[0269] Intranasal Delivery

[0270] Intranasal delivery occurs when particles are inhaled into the nasal cavity. Vaccine delivery through the nasal cavity increases drug bioavailability and lowers risk of systemic exposure. Intranasal drug administration is a non-invasive and convenient means to rapidly target vaccines of varying physical and chemical properties to the respiratory tract, including the nose and lungs. The vaccine or vaccine compositions of the present invention are intranasally delivered using any suitable delivery device known to the person skilled in the art, such as the aerosol delivery devices outlined above.

[0271] Antibiotic-inactivated

[0272] In the context of the vaccine or vaccine composition of the present invention, antibiotic- inactivated means that the bacteria is inactivated by an antibiotic. In embodiments, the antibiotic is a fluoroquinolone antibiotic, such as ciprofloxacin, or any other chemicals or expressing vectors which hinder the growth of microorganisms without substantially killing them. The bacteria inactivated by the fluoroquinolone antibiotic is not lysed and retains an intact morphology. Furthermore, the bacterial antigen structure remains intact, such that at least one or more bacterial antigens are not modified. This provides for an unexpected and enhanced immunogenicity compared to bacteria inactivated by other means such as heat treatment or chemical (e.g. aldehyde) treatment.

[0273] Furthermore, the antibiotic, is not limited to a quinolone or fluroquinolone antibiotic, and may be any antibiotic which permeates bacterial membrane and kills the bacteria by arresting cell division by inhibiting the function of DNA gyrase and / or DNA Topoisomerase IV. An inactivated vaccine is a vaccine consisting of virus particles, gram-positive or gramnegative bacteria (such as B. pertussis), or other pathogens or microorganisms that have been grown in culture and then killed to destroy disease-producing capacity. For example, the antibiotic (ciprofloxacin) in the culture medium is at an effective concentration hindering the growth of the B. pertussis without substantially killing the B. pertussis.

[0274] Antibacterial compounds

[0275] In the context of the vaccine or vaccine composition of the present invention, antibacterial compounds relate to compounds with antibacterial activity and particularly that inhibit bacterial division. Antibacterial means that the compound is capable of killing or causing the death of bacteria and / or inhibiting bacterial growth, including inhibiting growing bacterial cells. In a similar manner to the antibiotic, the antibacterial composition of the invention may also be a composition which targets essential bacterial enzymes involved in metabolic pathways, protein synthesis, DNA replication and repair, permeates bacterial membrane and / or kills the bacteria by arresting cell division by inhibiting the function of DNA gyrase and / or DNA Topoisomerase IV. Antibacterial compounds can include natural compounds or synthetic compounds. Natural antibacterial compounds include but are not limited to essential oils, lysozyme enzyme or bacteriocins. Certain essential oils, like tea tree oil or oregano oil, have been shown to possess antibacterial properties. These oils can disrupt bacterial membranes or interfere with their metabolism, potentially inhibiting division. Lysozyme is an enzyme is found in tears, saliva, and breast milk and can break down bacterial cell walls made of peptidoglycan. Bacteriocins are natural protein toxins produced by some bacteria that can kill or inhibit the growth of other bacteria. Synthetic compounds include but are not limited to antiseptics, disinfectants or metal nanoparticles. Antiseptic compounds are chemicals used to kill or inhibit the growth of microorganisms on surfaces or skin. Common examples include alcohol, chlorhexidine, and triclosan. Similar to antiseptics, disinfectants are stronger chemicals used to kill microorganisms on surfaces. Research suggests nanoparticles made of silver or copper can exhibit antibacterial properties. The exact mechanism may involve damaging bacterial membranes or interfering with their metabolism.

[0276] Disease associated with the bacteria

[0277] In the context of the present invention a disease associated with a bacteria means any disease that occurs as a result of infection with or exposure to the bacteria. For example, the disease associated with B. pertussis is pertussis disease, also commonly known as whooping cough. Other non-limiting examples, include S. aureus infections and diseases, from localized skin infections to severe systemic diseases, including but not limited to: skin and soft tissue infections and / or diseases, including boils, abscesses, cellulitis, impetigo, folliculitis and staphylococcal scalded skin syndrome (SSSS); bloodborne / bloodstream infections (bacteraemia), including sepsis; endocarditis; pneumonia; toxic shock syndrome; bone and joint infections, including osteomyelitis and septic arthritis.

[0278] These diseases / infections can be difficult to treat when present as methicillin-resistant Staphylococcus aureus (MRS A],

[0279] E. coll infections and diseases are also contemplated, for example to treat urinary tract infections (UTIs), pneumonia, meningitis, and severe invasive E. coll disease (IED), which can lead to sepsis and other bloodborne / bloodstream infections (bacteraemia).

[0280] Vector

[0281] In the context of the present invention, a vector is any expressing vector(s) transformed into bacteria that express antibiotic(s). The vector(s) could be DNA, RNAs (mRNA, tRNA etc.) or viral vector with antibiotic-expressing gene(s) cloned. In general, and throughout the specification, the term "vector" refers to a nucleic acid molecule to which it can transport another nucleic acid to which it is bound. Vectors include, but are not limited to, singlestranded, double-stranded, or partially double-stranded nucleic acid molecules; one or more free-ended, free-ended (eg, circular) nucleic acid molecules. Nucleic acid molecules containing DNA, RNA, or both; and various other polynucleotides known in the art. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which an additional DNA segment can be inserted, for example by standard molecular cloning techniques. Another type of vector is a viral vector in which the virus-derived DNA or RNA sequence is packaged into a virus (eg, retrovirus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, and adeno-associated virus).

[0282] Subject

[0283] As herein defined, a "subject” includes and encompasses mammals such as humans, primates and livestock animals (e.g. avians, sheep, pigs, cattle, horses, donkeys); laboratory test animals such as mice, rabbits, rats and guinea pigs; and companion animals such as dogs and cats. In a particular embodiment, the subject is a mammal, in particular a human. The term "subject” is interchangeable with the term "patient” as used herein.

[0284] Treatment / Therapy

[0285] The term "treatment” is used herein to refer to any regimen that can benefit a human or nonhuman animal. The term "treatment” and associated terms such as "treat” and "treating” means the reduction of the progression, severity and / or duration of a disease associated with a bacteria or infection with a bacteria or at least one symptom thereof, wherein said reduction or amelioration results from the administration of the vaccine composition of the present invention. The treatment may be in respect of bacterial infection and the treatment may be prophylactic (preventative treatment). Treatment may include curative or alleviative effects. Reference herein to "therapeutic" and "prophylactic" treatment is to be considered in its broadest context. The term "therapeutic" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylactic" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, therapeutic and / or prophylactic treatment includes amelioration of the symptoms of a bacterial infection or preventing or otherwise reducing the risk of developing a bacterial infection. The term "prophylactic" may be considered as reducing the severity or the onset of a particular condition. "Therapeutic" may also reduce the severity of an existing condition.

[0286] Pharmaceutical Compositions

[0287] Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to an active ingredient, a pharmaceutically acceptable excipient, carrier, buffer stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be, for example, oral, intravenous, intranasal or via oral or nasal inhalation. The formulation may be a liquid, for example, a physiologic salt solution containing non-phosphate buffer at pH 6.8-7.6, or a lyophilised or freeze-dried powder. Dose

[0288] The composition of the invention is typically administered to a subject in a "therapeutically effective amount”, this being an amount sufficient to show benefit to the subject to whom the composition is administered. The actual dose administered, and rate and time-course of administration, will depend on, and can be determined with due reference to, the nature and severity of the condition which is being treated, as well as factors such as the age, sex and weight of the subject being treated, as well as the route of administration. Further due consideration should be given to the properties of the composition, for example, its binding activity and in-vivo plasma life, the concentration of the antibody or binding member in the formulation, as well as the route, site and rate of delivery.

[0289] Dosage regimens can include a single administration of the vaccine composition, or multiple administrative doses of the vaccine composition. The vaccine compositions can further be administered sequentially or separately with other therapeutics and medicaments which are used for the treatment of the condition for which the composition of the present invention is being administered to treat. The vaccine of the present invention is administered in compositions, akin to the administration of subunit epitopes or antigens or therapeutics or antibodies to induce immunity, stimulate a therapeutic response and / or stimulate passive immunity. The quantity to be administered will vary for the patient (host) and condition being treated and will vary from one or a few to a few hundred or thousand micrograms, e.g., 1 pg to 1 mg, from about 100 ng / kg of body weight to 100 mg / kg of body weight per day and preferably will be from 10 pg / kg to 10 mg / kg per day. In embodiments, the vaccine or vaccine composition of the present invention is administered in a dose of between lxlO5to lxlO9CFU using a nebulizer or by intranasal administration at a dose of lxlO5to lxlO9CFU in 0.1-1.0 ml PBS. In embodiments, the vaccine or vaccine composition of the present invention is administered from a culture containing lxlO9CFU / ml. However, the actual dose of the composition administered, and rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and at the discretion of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Suitable regimes for initial administration and booster doses or for sequential administrations also are variable, and may include an initial administration followed by one, two or more subsequent administrations.

[0290] Gram-negative bacteria

[0291] "Gram-negative bacteria” generally refers to bacteria which produce a crystal violet stain that is decolorized in Gram staining, i. e. they do not retain crystal violet dye in the Gram staining protocol. As used herein, the term "Gram-negative bacteria” may describe without limitation one or more (i.e., one or a combination) of the following bacterial species: Acinetobacter baumannii, Acinetobacter haemolyticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Bacteroides fragilis, Bacteroides the ataioatamicron, Bacteroides distasonis, Bacteroides ovatus, Bacteroides vulgatus, Bordetella pertussis, Brucella melitensis, Burkholderia cepacia, Burkholderia pseudomallei, Burkholderia mallei, Prevotella corporis, Prevotella intermedia, Prevotella endodontalis, Porphyromonas asaccharolytica, Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Citrobacter freundii, Citrobacter koseri, Edwarsiella tarda, Eikenella corrodens, Enterobacter cloacae, Enterobacter aerogenes, Enterobacter agglomerans, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Haemophilus ducreyi, Helicobacter pylori, Kingella kingae, Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella rhinoscleromatis, Klebsiella ozaenae, Legionella penumophila, Moraxella catarrhalis, Morganella morganii, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Plesiomonas shigelloides, Proteus mirabilis, Proteus vulgaris, Proteus penneri, Proteus myxofaciens, Providencia stuartii, Providencia rettgeri, Providencia alcalifaciens, Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella typhi, Salmonella paratyphi, Serratia marcescens, Shigella flexneri, Shigella boydii, Shigella sonnei, Shigella dysenteriae, Stenotrophomonas maltophilia, Streptobacillus moniliformis, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Chlamydophila pneumoniae, Chlamydophila trachomatis, Ricketsia prowazekii, Coxiella burnetii, Ehrlichia chaffeensis, or Bartonella hensenae. The vaccines and vaccine compositions of the present invention will be useful in preventing or inhibiting pathogenic bacterial growth and in treating one or more bacterial infections, particularly but not necessarily exclusively involving Gram-negative bacteria and notably Bordetella pertussis. Gram-positive bacteria

[0292] "Gram-positive bacteria” can be identified by the presence of certain cell wall and / or cell membrane characteristics and / or by staining with Gram stain. Gram positive bacteria are known and can readily be identified and may be selected from but are not limited to the genera Actinomyces, Bacillus, Listeria, Lactococcus, Staphylococcus, Streptococcus, Enterococcus, Mycobacterium, Corynebacterium, and Clostridium and include any and all recognized or unrecognized species or strains thereof. Medically relevant species include Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. Bacillus species, which are spore-forming, cause anthrax and gastroenteritis. Spore-forming Clostridium species are responsible for botulism, tetanus, gas gangrene and pseudomembranous colitis. Corynebacterium species cause diphtheria, and Listeria species cause meningitis.

[0293] Acid-Fast Bacteria

[0294] Unlike traditional Gram-positive, or even Gram-negative bacteria, Mycobacterium tuberculosis is also classified as acid-fast due to its impermeable, mycolic acid-rich cell wall. Acid-Fast Bacteria include Tuberculosis complex M. tuberculosis, M. bovis), Leprosy-causing M. leprae)., Opportunistic nontuberculous mycobacteria M. kansasii, M. avium).

[0295] Antigen

[0296] As herein defined, an antigen is any substance that causes the immune system to produce antibodies against it. This means the immune system does not recognize the substance, and is trying to fight it off. An antigen may be a substance from the environment, such as chemicals, bacteria, viruses, or pollen. In the context of the vaccine composition of the present invention, a B. pertussis antigen is any immunogenic antigen from B. pertussis , for example pertussis toxin (PT), filamentous hemagglutinin (FHA), outer membrane proteins, fimbrial proteins 2 and 3 (FIM2 / 3), agglutinogens, adenylate cyclase, pertactin (PRN), or tracheal cytotoxin. S. aureus antigens include, but are not limited to, capsular polysaccharides (CP5 and CP8), the adhesion molecule ClfA, and enterotoxins. E. coli antigens colonization factor antigens (CFAs), heat-labile toxin (LT) from enterotoxigenic E. coli (ETEC), as well as Shiga toxin from enterohemorrhagic E. coli (EHEC). Fimbrial adhesins and siderophore receptors from uropathogenic E. coli (UPEC) can also be utilised. Additionally, components and effectors of the type III secretion system from enteropathogenic E. coli (EPEC) can be utilised. The vaccine of the present invention will have multiple antigens because the vaccine comprises the whole bacteria. The vaccine of the present invention will have at least one antigen from antibiotic-inactivated bacteria and could have hundreds or thousands of antigens from antibiotic-inactivated bacteria. These and other antigens when administered in a suitable formulation can generate immune responses that protect against bacterial disease and infection.

[0297] Mount / elicit / induce

[0298] As used herein, the terms "mount”, "mounted”, "elicit” "elicited”, 'induce’ or 'induced’ when used in relation to an immune response mean an immune response which is raised against the immunogenic determinant of a vaccine composition which is administered to a subject.

[0299] Immune Response

[0300] As used herein, the term "immune response" includes T cell mediated and / or B cell mediated immune responses that are influenced by modulation of T cell co-stimulation. The term immune response further includes immune responses that are indirectly effected by T cell activation such as antibody production (humoral responses) and the activation of cytokine responsive cells such as macrophages.

[0301] Thl-mediated Immune Response

[0302] A Thl-mediated immune response (or "Type 1 response") largely involves interaction with macrophages and CD8+ T cells and may be linked to interferon-y, TNF-p, interleukin-2 production. The Thl-mediated immune response promotes cellular immune system and maximizes the killing efficacy of the macrophages and the proliferation of cytotoxic CD8+T cells. The Thl-mediated immune response also promotes the production of opsonizing antibodies (e.g. IgG, IgM and IgA). The Type 1 cytokine IFN-y is driven by the production of interleukin- 12 by dendritic cells and macrophages, and via positive feedback, IL-12 stimulates the production of IFN-y in helper T cells, thereby promoting the Thl profile. Interferon-y also inhibits the production of cytokines such as interleukin-4, a cytokine associated with the Type 2 response, and thus it also acts to preserve its own response.

[0303] Thl7-mediated Immune Response

[0304] T helper type 17 (Thl7) cells are a distinct lineage of T cells that produce the effector molecules IL-17, IL-17F, IL-21, and IL-22. Although the role of Thl7 cells in autoimmunity is well documented, there is growing evidence that the Thl7 lineage and other interleukin (IL)- 17-producing cells are critical for host defence against bacterial, fungal, and viral infections at mucosal surfaces. IL-17-producing cells function as a bridge between innate and adaptive immunity against infectious diseases at the mucosa. CD4+ T helper cells (Th cells) are important mediators of adaptive immune responses. After interaction with antigen- presenting cells, T cells receive signals by engagement of the T-cell receptor (signal 1), costimulatory molecules (signal 2), and a complex network of cytokine signals (signal 3) and undergo activation and differentiation into effector CD4+ T cells. They assist B cells to make antibodies, activate the microbe killing capacity of macrophages and recruit other immune cells to infected or inflamed areas of the body.

[0305] Sterilising Immunity

[0306] Sterilising immunity means that the immune system is able to stop a pathogen, including viruses and bacteria, from replicating within your body. This typically happens when antibodies in the body are able to bind to the pathogen in places that prevent it from being able to enter a cell where it can start making copies of itself. B cells produce neutralising or sterilising antibodies, which are proteins that recognize specific proteins and structures on the surfaces of pathogens. Immune cells, including T cells, can also help to induce sterilising immunity either by killing virus-infected cells, promoting antibody production by B cells or recruiting and activating neutrophils and macrophages that take up and kill the pathogens. To achieve sterilising immunity, the subject’s needs to produce enough neutralising antibodies and T cells and it needs to be able to do so in the long term. Ideally, it leads to lifelong immunity.

[0307] The present invention will now be described with reference to the following examples which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention.

[0308] EXAMPLES

[0309] EXAMPLE 1

[0310] Materials and methods

[0311] Mice

[0312] C57BL / 6 mice were obtained from Charles River U.K. Mice were 6-10 weeks old at the initiation of experiments and housed in a specific pathogen-free facility in the Comparative Medicine Unit (CMU), Trinity College Dublin. All animal experiments were conducted according to the guidelines and under licenses approved by the Health Products Regulatory Authority (HPRA) of Ireland in accordance with prior ethical approval from Trinity College Dublin Animal Research Ethics Committee.

[0313] Antimicrobial activity of ciprofloxacin against B. pertussis

[0314] B. pertussis 338 from overnight culture in Stainer-Scholte (S&S) was adjusted to a concentration of 6* 106or 6* 107colony forming unit (CFU) / ml. Freshly prepared ciprofloxacin (Enzo Life Sciences Inc.) was diluted in nuclease-free water and added to the bacterial culture at concentrations of 0.1 to 0.5 mg / ml. The CFU / ml of live bacteria were quantified by performing CFU counts on Bordet-Gengou (BG) agar plates after 6, 12 and 24 h.

[0315] In vitro visualization of inactivated B. pertussis

[0316] B. pertussis 338 was inactivated with ciprofloxacin (0.25 and 0.5 mg / ml) for 24h followed by 4% PFA fixation (10 min room temperature). The samples were loaded into a Cytospin- MICROTEKNIK-JP-6 following the manufacturer's instructions and centrifuged at 2000 rpm for 10 min. B. pertussis 338 was detected using anti- / / . perlussis-LC) - / 8 (Thermo Fisher, clone D26E) and goat anti-mouse IgG3-Alexa Fluor 594 (Life Technologies) antibodies. Image acquisition was performed using an SP8 confocal microscope (Leica, Germany).

[0317] Vaccines and immunization

[0318] B. pertussis 338 from overnight culture was adjusted to 6 / I 07CFU / ml in S&S medium and treated with 0.25 mg / ml ciprofloxacin. After 3 h of antibiotic treatment (37°C, 180 rpm) the bacteria were collected by centrifuging at 3000 rpm for 20min at 4°C and washed 2X with 1% casein solution. The concentration of the collected bacteria was adjusted to 1 x 109CFU / ml. To confirm inactivation of the bacteria, 100 pl (3x) of antibiotic-inactivated B. pertussis (AIBP) were plated on BG agar. The plates were monitored for any live bacterium after 3-5 days. Mice were immunized once or twice after a 4 week interval by aerosol administration of AIBP using a nebulizer (PARI TurboBOY SX) from a culture at 1 x 109CFU / mL over 10 min as described previously

[0035] or intranasally (i.n.) as described previously

[0036] by placing two 15 pl droplets of the AIBP vaccine [3xl05, 3xl06or 3xl07CFU / dose) on the mouse nares or by intramuscularly (i.m.) administration of 1 / 50 human dose of a commercial aP vaccine (Boostrix, GlaxoSmithKline) or wP vaccine (NIBSC code: 94 / 532).

[0319] To confirm that there are no live bacteria in the AIBP vaccine, CFU counts were performed on freshly prepared AIBP vaccine and digested nasal tissue or lung homogenates on three mice 2 h and 3 days after vaccination

[0037] , The plates were monitored for any live bacterium after 3- 5 days.

[0320] B. pertussis culture and respiratory challenge

[0321] B. pertussis 338 bacteria were grown from frozen stocks for 3 days on BG plates. Bacteria were then collected and cultured in supplemented S&S medium overnight at 37°C in a shaking incubator at 180 rpm. Bacteria were centrifuged and resuspended in 1% casein solution, and the OD was measured at 600 nm

[0037] , Mice were infected by the aerosol challenge administered using a nebulizer from a culture at 1 x 109CFU / mL over 10 min as described previously

[0035] or i.n. with l * 109CFU / mL of live B. pertussis. In some experiments, immunized mice were compared with convalescent mice. Convalescent mice were defined as mice that were >60 days post B. pertussis challenge on the day of the second immunization for vaccinated mice.

[0322] Immunohistofluorescence microscopy of frozen tissue sections

[0323] Tissues were removed from mice 24 h post infection with B. pertussis 338 or aerosol immunization with AIBP and fixed with 4% PFA for 24h at 4 °C. Samples were dehydrated in 30% sucrose for 24 h, embedded in OCT, and snap frozen in dry ice. B. pertussis was detected in 15 pm tissue sections using anti- / / . perlussis- O - (ThermoFisher, clone D26E), goat antimouse IgG3-Alexa Fluor 594 (Life Technologies) antibodies and ActinGreen™ 488 Ready Probes. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Image acquisition was performed using an SP8 confocal microscope (Leica, Germany). Isolation of lung and nasal tissues

[0324] Whole lung and nasal tissue (including the nasal cavity and nasal turbinates) were aseptically removed and transferred to petri dish and chopped with a scalpel. Lungs and nasal tissues were digested in PBS containing collagenase D (1 mg / mL; Sigma- Aldrich) and DNAse I (20 U / ml; Sigma-Aldrich) for Ih at 37°C. Cells were centrifuged at 1350 rpm and supernatants were used to assess CFU counts. Lungs and nasal tissues were mashed through 70 pm strainer, cells were centrifuged at 1350 rpm, and used for flow cytometric analysis.

[0325] Inflammatory responses induced by AIBP vaccine

[0326] Mice were immunized by aerosol administration with 1 * 109CFU / ml AIBP, i.m. administration of a wP vaccine (1 / 50 human dose) or infected with P I O9CFU / mL live B. pertussis. Mice were euthanized 4 h or 24 h after administration. Serum, lung homogenate and nasal tissue supernatants were collected for analysis of inflammatory cytokines (IL-6, IL-ip, IL-12, IL-23, TNF) by ELISA and cells were used for cell activation using flow cytometric analysis.

[0327] Bone marrow derived dendritic cells (BMDC) isolation and in vitro stimulation

[0328] Murine BMDCs were prepared as described previously

[0027] , BMDCs (l x 106) in RPMI 1640 medium (without penicillin and streptomycin) were cultured in 96-well U-bottom plates with AIBP, live B. pertussis 338 or heat killed B. pertussis (HKBP) at a bacterium -to-cell ratio of 10: 1 for 24h at 37°C in a 5% CO2 incubator. MHC class II and co-stimulatory molecule expression was evaluated using flow cytometric analysis. Supernatants were collected from cell cultures for quantification of T cell polarizing cytokines by ELISA.

[0329] Quantification of TRM cells

[0330] To discriminate circulating from tissue-resident lymphocytes by flow cytometry, mice were injected i.v. with 1.5 pg of PE-conjugated anti-CD45 antibody (30-F11; eBioscience) in 200 pl PBS 10 min prior to euthanasia. Circulating lymphocytes are labeled CD45 i.v.+, while tissueresident lymphocytes are protected from i.v. labeling, are therefore identified as CD45 i.v.'. Mononuclear cell suspensions from lungs or nasal tissue were incubated with LIVE / DEAD Aqua (Invitrogen). The cells were incubated with Fc block (BD Biosciences) (1 :50) to block IgG Fc receptors followed by a surface staining with fluorochrome-conjugated anti-mouse antibodies specific for CD69 (cloneH1.2F3), CD45R / B220 (clone RA3-6B2), CD8 (clone53- 6.7), CD3 (clone 145-2C11), CD4 (clone GK1.5), CD44 (clone IM7), CD62L (cloneMEL-14), CD103 (clone M290), MHC class II (MHCII; clone M5 / 114.15.2), CD80 (clone 16-10Al) and CD86 (clone GL-1) from Biolegend, BD Biosciences or Invitrogen and fixed with 2% paraformaldehyde (PF A, Thermo Fisher Scientific). TRM cells were defined through lack of in vivo labelling with anti-CD45. Cells that were CD45i.v. CD44+CD62L' and express CD69, with or without CD 103 were considered to be TRM cells.

[0331] For detection of cytokine producing B. pertussis-s^Qc c, T cells, mononuclear cells were stimulated for 16 hours with HKBp (105CFU / mL), anti-CD28 and anti-CD49d (Ipg / mL; BD Biosciences). Brefeldin A (5pg / ml) was added for the final 4 hours of culture. The cells were fixed, permeabilized and stained using the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions using antimouse antibodies specific for IFN-y (XMG1.2) or IL- 17 (clone TCI 1-18H10). Flow cytometric analysis was performed on an Aurora, and data were acquired using SpectroFlo. Data were analysed using FlowJo software (Tree Star).

[0332] IgGl, IgG2c and IgA in serum and nasal associated lymphoid tissue (NALT)

[0333] Serum samples were prepared by centrifugation of whole blood at 5,000*g at 4°C for 10 min after bleeding of mice by cardiac puncture. NALTs were dissected from euthanized mice and immediately cultured in complete RPMI supplemented with antibiotics (penicillin 1%, streptomycin 1%, Amphotericin B 1 / 1000 and Geneticin 1 / 1000) and sonicated B. pertussis (sBP; 5pg / ml). After 72h, the supernatants were collected and stored at -20°C. B. pertussis- specific antibodies in individual mouse sera and NALT culture supernatants were quantified by ELISA using plate-bound HKBp (107CFU / ml) and biotin-conjugated anti-mouse IgGl, IgG2c or IgA (1 :1500 HRP-conjugated, Southern Biotech). The reaction was developed using TMB and 1 M H2SO4 and the plates were read using a SpectraMax ABS microplate reader (Molecular Devices) at 450 nm.

[0334] Statistical analysis

[0335] All statistical analyzes were performed using Graph-Pad Prism 9.0 Software. Data were presented as mean ± SEM. Data were analyzed by one-way ANOVAs or two-way ANOVAs followed by post hoc Tukey’s test for multiple comparisons. P values <0.05 were considered significant. Experiment 1: Effect of ciprofloxacin on survival and morphology of B. pertussis in culture

[0336] Ciprofloxacin is a broad-spectrum antibiotic of the fluoroquinolone drug class against both Gram-positive and Gram-negative bacteria. Ciprofloxacin acts by inhibiting bacterial DNA synthesis, either by rapidly inhibiting the activity of DNA gyrase or by inhibiting the activity of DNA Topoisomerase IV, leading to bacterial cell death

[0028] ,

[0337] The inventors tested the effect of ciprofloxacin on the growth of B. pertussis 388 in liquid culture. B. pertussis were cultured overnight in supplemented S&S medium and the concentration adjusted to 6xl06or 6xl07CFU / ml and treated with 0.1 - 0.5 mg / ml of ciprofloxacin. The results demonstrate that 0.1 mg / ml of ciprofloxacin completely inactivated B. pertussis at an initial concentration of 6xl06CFU / ml as early as 2h (Figure 1A). Furthermore, 0.25 mg / ml of ciprofloxacin inactivated B. pertussis at an initial concentration of 6xl07CFU / ml by 2h (Figure IB). Fluorescence microscopic analysis ofB. pertussis showed that the length of bacteria increased up to 9.5 pm 24 h after inactivation with 0.25 or 0.5 mg / ml ciprofloxacin (Figure 2A and B).

[0338] Experiment 2: The AIBP vaccine promotes BMDCs maturation

[0339] BMDCs were cultured with AIBP, live B. pertussis or HKBp (bacterium-to-cell ratio of 10:1). MHC class II and co -stimulatory molecule expression were evaluated using flow cytometric analysis 24 h after stimulation. Similar to stimulation with live B. pertussis, AIBP significantly upregulated the expression of MHCII, CD80 and CD86 when compared to HKBp (Figure 3). The enhancement of MHCII and CD86 with AIBP was greater than that induced with live B. pertussis and significantly greater than that induced with HKBp.

[0340] Analysis of inflammatory cytokines in collected supernatants by ELISA showed that stimulation of BMDCs with AIBP, live B. pertussis or HKBp significantly increased the concentration of Thl7-polarizing cytokines IL-lp, IL-6, IL-23 and TNF and Thl-promoting cytokine IL-12 when compared to unstimulated control (Figure 4). These results demonstrate that the AIBP vaccine promotes DC maturation. Experiment 3: Aerosol administration of AIBP promotes T cell polarizing cytokines in the local but not systemic inflammatory responses

[0341] The inventors first demonstrate that B. pertussis could be detected in the lung by immunohistochemistry 24 h after aerosol administration of live B. pertussis (Figure 5 A) or ciprofloxacin-inactivated B. pertussis (Figure 5B). The results demonstrate B. pertussis are present in the lung 24 h after infection or vaccination.

[0342] The inventors next assessed local and systemic inflammatory responses induced with AIBP compared with a wP vaccine or infection with live virulent B. pertussis. Mice were aerosol immunized with the AIBP vaccine or live virulent B. pertussis or immunized i.m. with a wP vaccine or PBS. The concentration of inflammatory cytokines were assessed in lungs, nasal tissue and serum 4 and 24 h after administration. Similar to aerosol challenge with live B. pertussis or i.m. immunization with a wP vaccine, aerosol immunization of the mice with AIBP significantly increased the concentrations of Thl7-polarizing cytokines IL-1 p, IL-6, IL-23 and TNF in the lungs 4 hours after administration (Figure 6). These increases were transient and had returned to baseline after 24 hours. The induction of T cell polarizing cytokines was less dramatic in the nasal tissue. However, aerosol delivery of the AIBP vaccine or live B. pertussis, but not the wP vaccine induce significant IL-6 production in the nasal tissue 4 hours after administration (Figure 7).

[0343] The concentrations of IL-6, IL-1 , IL-12, IL-23 and TNF were enhanced in the serum of mice 4 hours after i.m. immunization with the wP vaccine (Figure 8). In contrast, aerosol administration of the AIBP vaccine or live B. pertussis did not significantly increase inflammatory cytokine concentrations in the serum over that detected in mice immunized with PBS (Figure 8). These findings are consistent with the data on in vitro stimulation of BMDCs and suggest that the AIBP vaccine transiently induces production of T cell polarizing cytokines in the respiratory tissue, but unlike the wP vaccine, this does not extend to a systemic inflammatory response. Experiment 4: Aerosol immunization of mice with AIBP vaccine protects mice against nasal and lung infection with B. pertussis

[0344] The inventors next examined the immunogenicity and protective efficacy of the AIBP vaccine in vivo. B. pertussis from overnight culture was adjusted to 6xl07CFU / ml and treated with 0.25 mg / ml ciprofloxacin. After 3 h of antibiotic treatment the concentration of the collected bacteria was adjusted to lxlO9CFU / ml. Mice were immunized one or twice (4 week interval) by aerosol inoculation with AIBP from a culture equivalent to lxlO9CFU / ml or with aP vaccine (1 / 50 the human dose) twice by i.m. route. Mice were challenged by aerosol exposure to virulent B. pertussis 6 weeks after the first immunization.

[0345] Immunization AIBP vaccine conferred a high level of protection against lung and nasal infection with B. pertussis (Figure 9). A single dose of the AIBP vaccine conferred similar protection in the lung to two doses of the current aP vaccine (Figure 9A) and bacteria were completely cleared from the lungs 14 days after challenge of mice immunized with 2 doses of the AIBP vaccine. A single dose of AIBP vaccine also conferred significant protection against infection of the nasal tract, whereas mice immunized with two doses of the AIBP vaccine had completely cleared the infection from the nose 14 days after challenge (Figure 9B). In contrast, immunization with the aP vaccine by the i.m. route did not confer any protection against nasal infection with B. pertussis with similar bacterial loads in unimmunized control mice (Figure 9B). These findings demonstrate that the aerosol-delivered AIBP vaccine, even a single dose, is highly effective in preventing infection of the lung and nasal cavity with B. pertussis.

[0346] Experiment 5: Immunization with the AIBP vaccine induces IL-17 and IFN-y-secreting CD4 cells in the lung and nasal tissue

[0347] The inventors analysed T cell responses in the respiratory tract of mice immunized with the AIBP or aP vaccines prior to challenge with live B. pertussis using flow cytometry analysis of ex vivo antigen stimulated immune cells from the lungs and nasal tissues. A significant number of CD4 TRM cells were detected in the lungs (Figure 10A) and nose (Figure 10B) after a single immunization with the AIBP vaccine and this was significantly enhanced following a booster immunization with the same vaccines. In contrast, CD4 TRM were not enhanced over the PBS-immunized control in mice immunized with the aP vaccines (Figure 10). Analysis of T cell response in the respiratory tract 7 days after live B. pertussis challenge confirmed that one dose of the AIBP vaccine induced significant number of CD4 TRM in the lungs (Figure 11 A) and nasal tissue (Figure 11B), which was augmented in mice that had received two doses of the same vaccine. In contrast CD4 TRM were at the same frequency as in the non-immunized control in mice immunized with the aP vaccine (Figure 11A, B). These findings demonstrate that immunization of mice with one or two doses of AIBP vaccine induced CD4 TRM cells in the respiratory tract, whereas the current parenterally- administered aP vaccine does not.

[0348] The inventors assessed cytokine production by respiratory CD4 cell by intracellular cytokine staining of lung or nasal tissue immune cells with HKBp before and 7 days after challenge with B. pertussis. The results demonstrate that B. pertussis-specific IL-17- and IFN-y- producing CD4 cells (Figure 12A, Figure 12B and Figure 13A, Figure 13B) were induced in nasal tissues and IL-17 producing CD4 cells were induced in lung tissues of mice immunized with two doses of the AIBP vaccine (Figure 12C, Figure 12D and Figure 13C, Figure 13D). Weaker cytokine-secreting CD4 cells were also detected after a single immunization with the AIBP vaccine.

[0349] Experiment 6: Immunization with two dose of AIBP vaccine generate IgGl, IgG2c and IgA responses in serum and NALT

[0350] The inventors quantified B. pertussis-specific antibody response by ELISA in the serum and supernatant of antigen-stimulated NALT cells from immunized mice. The results demonstrate that immunization of mice with two doses of AIBP vaccine induces potent IgGl, IgG2c in serum, which was stronger than that induced with the aP vaccine (Figure 14A, Figure 14B). Immunization with the single dose of AIBP vaccine failed to induce detectable antibodies in serum or NALT (Figure 14A, Figure 14B and Figure 14C). Two doses of the AIBP vaccine also induced high levels of IgA detectable in the supernatant of NALTs culture (Figure 14C). In contrast IgA was undatable after two doses of the aP vaccine. These findings demonstrate that the AIBP vaccine induced potent B. pertussis-specific IgG in serum and IgA in the nasal mucosa, but this requires a primary and booster immunization.

[0351] Modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art, without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

[0352] Experiment 7 : Protective immunity induced with the AIBP vaccine is more effective than that generated by previous infection or the wP vaccine.

[0353] Good quality wP vaccine are considered to be the gold standard for pertussis vaccine in terms of efficacy, but not safety. Furthermore, immunity induced by previous infection is thought to surpass any licensed pertussis vaccine. The present inventors compared the protective efficacy of two doses of the wP vaccine, administered by the standard i.m. route, with two doses of the AIBP vaccine, delivered to the respiratory tract by aerosol. Mice were challenge with live B. pertussis at 6 weeks after the two immunization at 0 and 4 weeks or 12 weeks after previous infection. The data demonstrate that the AIBP vaccine, the wP vaccine and previous infection all conferred complete protection against B. pertussis infection of the lungs (Figure 15). The wP vaccine also induced a high level of protection in the nose tract, but bacteria were still detectable in the nose 14 and 21 day post live B. pertussis challenge. The protection induced by previous infection was somewhat better, with complete clearance from the nose by day 21. However, the best protection was observed with the aerosol- delivered AIBP vaccine of the present invention; these mice had completely cleared the infection from the nose by day 14. These findings demonstrate that protective immunity induced with the AIBP vaccine of the present invention is superior to that induced by previous infection or immunization with the wP vaccine.

[0354] Assessment of antibody responses demonstrated that the wP vaccine failed to induce IgA, whereas two doses of the AIBP vaccine or previous infection induced B. pertussis-specific IgA in NALT (Figure 16A). The wP vaccine induce substantial B. pertussis-specific IgGl in the serum, whereas low levels of B. pertussis-specific IgGl were detected in the serum after immunization with the AIBP vaccine (Figure 16B). Substantial quantities B. pertussis-specific IgG2c was induced with two doses of the wP vaccine and lower levels after previous infection or immunization with the AIBP vaccine (Figure 16C). These findings demonstrate that the serum antibody responses induced with AIBP are weaker than those induced with the wP vaccine or previous infection, but unlike the wP vaccine the AIBP vaccine, line natural infection, induces mucosal IgA. The inventors assessed B. pertussis-specific T cell responses in the lymph nodes (LN) of immunized mice. LN cells were stimulated with heat-killed B. pertussis (HKBP) and after 3 days cytokines in supernatants were quantified by ELISA. LN cells from mice immunized with two doses of the AIBP vaccine produced substantial quantities of IL-17 and IFN-y. The IL-17 production was similar to that induced by previous infection and significantly greater than that induced by two doses of the wP vaccine (Figure 17). The findings demonstrate that the AIBP vaccine of the present invention is more effective than the wP vaccine or previous infection at induing B. pertussis-specific Thl and Thl7 responses in mice.

[0355] Assessment of CD4 TRM cells in the respiratory tract by flow cytometry demonstrated that there was significant accumulation of CD4 TRM cells in the lungs of mice immunized with the AIBP vaccine and this was significantly stronger than that induced by previous infection or immunization with the wP vaccine (Figure 18A-B). In contrast, accumulation of CD4 TRM in the nasal tissue was strongest in convalescent mice. However, the numbers of CD4 TRM cells were significantly higher in mice immunized with the AIBP compared with the wP vaccine.

[0356] Assessment of cytokine secreting B. pertussis-specific CD4 cells demonstrated that IL-17- secreting or IFN-y-secreting B. pertussis-specific CD4 were significantly higher in the lungs of mice immunized with the AIBP vaccine compared with the wP vaccine or previous infection (Figure 18 C-D). The AIBP vaccine, like natural infection, also promote accumulation of IL-17- secreting or IFN-y-secreting B. pertussis-specific CD4 in the nasal tissue and this was greater than that generated with the wP vaccine (Figure 18 E-F). These findings demonstrate the AIBP vaccine of the present invention is more effective than the wP vaccine and similar to previous infection, at inducing B. pertussis-specific Thl and Thl7-like CD4 cells in the respiratory tissue.

[0357] Experiment 8: Immunization with the AIBP vaccines overcomes defects in the current aP vaccine

[0358] The existing aP vaccine does not induce B. pertussis-specific Thl or Thl7 cells or respiratory TRM cells or prevent infection of nasal tract with B. pertussis and there is some evidence that it may suppress induction of CD4 TRM cells and compromise bacterial clearance post live B. pertussis challenge. The inventors examined that capacity of the AIBP vaccine of the present invention to overcome the deficits and suppressive effects of the aP vaccine. Mice were immunized twice (0 and 4 weeks) with the aP vaccine and boosted once (8 weeks) or twice (8 and 12 weeks) with AIBP or given the AIBP (once or twice) only and challenged by aerosol with B. pertussis at week 14. The data show that the bacterial load in the lungs was reduced to a similar extent after B. pertussis challenge of mice immunization with one dose of AIBP compared with two doses of the aP vaccine (Figure 19A). Immunization with two doses of the AIBP vaccine resulted in complete clearance of bacteria from the lungs by day 7, and this was not affected by prior immunization twice with the aP vaccine.

[0359] Immunization with the aP vaccine twice had no impact on infection of the nasal cavity (Figure 19B). In contrast, a single immunization with the AIBP vaccine significantly reduced the bacterial load in the nasal cavity and two immunizations resulted in complete clearance by day 14 post live B. pertussis challenge. Prior immunization with two doses of the aP vaccine had no impact on the protective efficacy of one or two doses of the AIBP vaccine. These findings demonstrate that the AIBP vaccine is highly effective in preventing lung and nasal infection with B. pertussis and this is not compromised by prior immunization with the aP vaccine.

[0360] Assessment of antibody responses demonstrated that the aP vaccine failed to induce IgA, whereas two doses of the AIBP vaccine induced B. pertussis-specific IgA in NALT and this was not affected by prior immunization with the aP vaccine (Figure 20A). Two doses of the aP vaccine induce substantial B. pertussis-specific IgGl in the serum and this was not enhanced by one or two booster doses with the AIBP vaccine (Figure 20B). Low levels of B. pertussisspecific IgGl were detected in the serum after two doses of the AIBP vaccine, but IgGl was at background levels in the serum of mice immunization one dose of the AIBP vaccine (Figure 2 OB). Substantial quantities B. pertussis-specific IgG2c was induced with two doses of the AIBP vaccine (Figure 20C). In contrast IgG2c responses were close to background in mice immunized with two doses of the aP vaccine or one dose of the AIBP vaccine. However, IgG2c was induced in mice primed with two doses of the aP vaccine and boosted with one or two doses of AIBP. These finding confirm previous reports that the aP vaccine fails to induce IgA or IgG2c, which is considered to be the opsonizing antibody driven by Thl responses, but instead induces IgGl, which has been associated with Th2 responses. In contrast the AIBP vaccine induced mucosal IgA and serum IgG2c and this is not reduced by prior immunization with the aP vaccine. The inventors assessed B. pertussis-specific T cell responses in the LN of immunized mice. LN cells were stimulated with HKBp and after 3 days cytokines in supernatants were quantified by ELISA. Immunization (twice) with the aP vaccine failed to induce IL-17 or IFN-y producing B. pertussis-specific cells in the LN (Figure 21). In contrast, LN cells from mice immunized with one or two doses of the AIBP vaccine produced substantial quantities of IL-17 and IFN- y and this was not significantly affected by prior immunization with two doses of the aP vaccine. The findings demonstrate that boosting with the AIBP vaccine is able to induce B. pertussis-specific Thl and Thl7 cells in mice previously primed with the Th2-inducing aP vaccine.

[0361] Assessment of CD4 TRM cells in the respiratory tract by flow cytometry demonstrated that CD4 TRM cells were at background levels in mice immunization with the aP vaccine (Figure

[0362] 22). In contrast, there was significant accumulation of CD4 TRM cells in the lungs and nasal tissue of mice immunized once with the AIBP and this was augmented further following a booster dose with the AIBP vaccines. Prior immunization with two doses of the aP vaccines did not affect the ability of one or two doses of the AIBP vaccine to induce CD4 TRM cells.

[0363] Assessment of cytokine secreting B. pertussis-specific CD4 cells demonstrated that the aP vaccine failed to generate B. pertussis-specific CD4 cells in the lungs or nasal tissue (Figure

[0364] 23). In contrast, IL-17 and / or IFN-y secreting B. pertussis-specific CD4 were significantly augmented in the lungs and nasal tissue of mice immunized once or twice with the AIBP vaccine (Figure 23). These responses were not significantly affected by prior immunization with the aP vaccine. The discovery of the inventors demonstrates that the AIBP vaccine of the present invention induces IL-17 and IFN-y-secreting B. pertussis-specific CD4 cells in the respiratory tract and shows that prior immunization with aP vaccines does not suppress the induction of Thl and Thl7-type CD4 cells with the AIBP vaccine.

[0365] Experiment 9: Intranasal delivery of the AIBP vaccines is highly effective at preventing lung and nasal infection with B. pertussis.

[0366] The inventors have demonstrated that aerosol delivery of the AIBP is highly immunogenic and confers perfection against lung and nasal infection with B. pertussis. Furthermore, the efficacy of the AIBP vaccine was greater than that generated with either aP or wP vaccines. The inventors next examined whether intranasal (i.n.) rather than aerosol delivery of the AIBP was also effective and if the efficacy was equal to or greater than previous infection, which is considered to the most effective method of inducing protective immunity.

[0367] Mice were immunized twice (0 and 4 weeks) with the AIBP vaccine i.n. by placing two 15 pl droplets of the AIBP vaccine (low, medium or high doses; 3xl05, 3xl06or 3xl07CFU / dose) on the mouse nares. Alternative mice were infected by i.n. inoculation with live B. pertussis. Mice were challenged by aerosol with B. pertussis at week 6. The bacterial load was assessed in respiratory tissue 2 hr and 7 days after B. pertussis challenge. Two hours after challenge the number of CFU in the lungs and nasal tract was similar in all groups (Figure 24), demonstrating the consistency of the challenge and that all immunization group received a similar challenge dose. Seven days after challenge, the control mice immunized with PBS had high bacterial counts in the lungs and nose. In contrast, mice immunized i.n. with the high dose of the AIBP vaccine or had previously been infected (convalescent mice) had no detectable bacteria in the lung 7 days after challenge (Figure 24A). The bacterial load in the lungs was also substantially reduced in mice immunized with medium and low doses of the AIBP vaccine. The AIBP vaccine also significantly reduced the bacterial load in the nose (Figure 24B). The highest level of protection in the nose was observed with the high dose of the AIBP and this more effective than previous infection. The medium and dose of the AIBP vaccine delivered by the i.n. route also substantially reduced the B. pertussis infection of the nose.

[0368] Assessment of antibody responses demonstrated that immunization with the high dose of the AIBP vaccine induced B. pertussis-specific IgA in NALT (Figure 25 A). The high medium and low doses of the AIBP vaccine induced B. pertussis-specific IgGl and Ig2a in the serum (Figure 25 B and C). Immunization with the high dose of the AIBP vaccine induced B. pertussisspecific Ig2a, which was greater than that induced by previous infection (Figure 25 B).

[0369] Assessment of CD4 TRM cells in the respiratory tract by flow cytometry demonstrated significant accumulation of CD4 TRM cells in the nasal tissue of mice immunized i.n. with the AIBP vaccine. Furthermore, the number of CD4 TRM cells in nasal tissue was higher in mice immunized with the high dose of the AIBP vaccine when compared with mice previously infected (convalescent) (Figure 26A). Intranasal delivery of the AIBP vaccine did not increase the number of CD4 TRM cells in the lungs (Figure 26B), suggesting that i.n. route favours induction of T cell responses in nasal tissue. Assessment of cytokine secreting B. pertussis-specific CD4 cells demonstrated that IL-17 and / or IFN-y secreting B. pertussis-specific CD4 were significantly augmented in the lungs and nasal tissue of mice immunized i.n with the high dose of the AIBP vaccine (Figure 27). These responses were similar to those induced by previous infection. IL-17 and / or IFN-y secreting B. pertussis-specific CD4 were also detected in the nose following immunization with a medium dose of the AIBP vaccine (Figure 27).

[0370] These findings demonstrate that like the aerosol delivered AIBP vaccine, the intranasal delivered AIBP vaccine is highly immunogenic, inducing local Thl and Thl7 type T cells and IgA in the nasal tract and confers protection against lung and nasal infection with B. pertussis.

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[0409] EXAMPLE 2

[0410] Materials and methods

[0411] Antimicrobial activity of levofloxacin and chloramphenicol against B. pertussis

[0412] B. pertussis 338 from overnight culture in Stainer-Scholte (S&S) was adjusted at a starting concentration of 6xl07colony forming unit (CFU) / ml. Freshly prepared levofloxacin (Enzo Life Sciences Inc.) and chloramphenicol (Thermoscientific) at concentrations of 0.25 to 3 mg / ml and 25 to 100 pg / ml, respectively, were diluted in nuclease-free water and added into the bacterial culture. The CFU / ml of live bacteria were quantified by performing CFU counts on Bordet-Gengou (BG) agar plates after 3, 6, 12 and 24 h.

[0413] Antimicrobial activity of ciprofloxacin and levofloxacin against S. aureus

[0414] S. aureus (Strain Newman) from 3h culture in Nutrient broth medium was adjusted at a starting concentration of 3xlO10colony forming unit (CFU) / ml. Freshly prepared ciprofloxacin or levo floxacin (Enzo Life Sciences Inc.) at concentrations of 0.1 to 3 mg / ml were diluted in nuclease-free water and added into the bacterial culture. The CFU / ml of live bacteria were quantified by performing CFU counts on Nutrient agar plates after 3, 6, 12 and 24 h.

[0415] Imaging of inactivated B. pertussis

[0416] B. pertussis 338 was inactivated with 1 mg / ml levofloxacin or 100 pg / ml chloramphenicol for 24h followed by 4% PFA fixation (10 min room temperature). B. pertussis was heat killed (HKBp) by placing it in a heat block set at 95°C for 30 minutes. The samples were loaded into a Cytospin-MICROTEKNIK-JP-6 following the manufacturer's instructions and centrifuged at 2000 rpm for 10 min. B. pertussis 338 was detected using anti-B. pertussis-LOS-A. (Thermo Fisher, clone D26E) and goat anti-mouse IgG3-Alexa Fluor 594 (Life Technologies) antibodies. Image acquisition was performed using an SP8 confocal microscope (Leica, Germany).

[0417] Imaging of inactivated E.col Top 10

[0418] E. coli top 10 was inactivated with 1 mg / ml levofloxacin, 0.25mg / ml ciprofloxacin, 100 pg / ml streptomycin (Sigma-Aldrich) and 50 pg / mL kanamycin (fluorochem), for 24h followed by 4% PFA fixation (10 min room temperature). The samples were loaded into a Cytospin- MICROTEKNIK-JP-6 following the manufacturer's instructions and centrifuged at 2000 rpm for 10 min. E. coli Top 10 was detected using FM™ 4-64 dye according to the manufacturer’s instructions. Image acquisition was performed using an SP8 confocal microscope (Leica, Germany).

[0419] Imaging of inactivated S. aureus

[0420] S. aureus (Strain Newman) was inactivated with lmg / ml levofloxacin, lmg / ml ciprofloxacin, lOOpg / mL penicillin (Sigma-Aldrich) or 100 pg / mL streptomycin (Sigma-Aldrich) for 24h followed by 4% PFA fixation (10 min room temperature). The samples were loaded into a Cytospin-MICROTEKNIK-JP-6 following the manufacturer's instructions and centrifuged at 2000 rpm for 10 min. S. aureus was detected using FM™ 4-64 dye according to the manufacturer’s instructions. Image acquisition was performed using an SP8 confocal microscope (Leica, Germany). Antibiotic inactivation of B. pertussis 338 and S. aureus for immunogenicity testing

[0421] B. pertussis 338 from overnight culture was adjusted to 6xl07CFU / ml in S&S medium and treated with 0.25 mg / ml ciprofloxacin for 3 and 24 hours, 1 mg / ml levo floxacin for 3 hours, or 100 pg / ml chloramphenicol for 3 hours.

[0422] S. aureus (Strain Newman} from 3 h our culture in Nutrient broth medium was adjusted to a starting concentration of 3xlO10CFU / ml and treated with 1 mg / ml levofloxacin for 3 hours.

[0423] Mice

[0424] C57BL / 6 mice were obtained from Charles River U.K. Mice were 6-10 weeks old at the initiation of experiments and housed in a specific pathogen-free facility in the Comparative Medicine Unit (CMU], Trinity College Dublin. All animal experiments were conducted according to the guidelines and under licenses approved by the Health Products Regulatory Authority (HPRA] of Ireland in accordance with prior ethical approval from Trinity College Dublin Animal Research Ethics Committee.

[0425] Vaccines and immunization

[0426] After antibiotic treatment (37QC, 180 rpm] the bacteria were collected by centrifuging, at 3000 rpm for 20 min at 4 C and washed twice with 1% casein solution. The concentration of the collected bacteria was adjusted to lxlO9CFU / ml. To confirm inactivation of the bacteria, 100 pl (3x] of antibiotic-inactivated B. pertussis (AIBP] or antibiotic-inactivated S. aureus were plated on BG agar and Nutrient agar, respectively. The plates were monitored for any live bacteria after 1-5 days. Mice were immunized once using a nebulizer (PARI TurboBOY SX] from a culture at lxlO9CFU / mL over 10 min as described previously [1], To confirm that there are no live bacteria in the antibiotic inactivated vaccine, CFU counts were performed on digested nasal tissue or lung homogenates on three mice 2 hours and 3 days after vaccination.

[0427] Isolation of lung and nasal tissues

[0428] Whole lung and nasal tissue (including the nasal cavity and nasal turbinates] were aseptically removed and transferred to petri dish and chopped with a scalpel. Lungs and nasal tissues were digested in PBS containing collagenase D (1 mg / mL; Sigma-Aldrich] and DNAse I (20 U / ml; Sigma-Aldrich] for lh at 37°C. Cells were centrifuged at 1350 rpm and supernatants were used to assess for any live bacteria. Lungs and nasal tissues were mashed through 70 pm strainer, cells were centrifuged at 1350 rpm, and used for flow cytometric analysis. Quantification of pertussis toxin (PT)

[0429] Mice were immunized by aerosol administration of the AIBP vaccine (at a concentration equivalent to lxlO9CFU / ml), or infected with live B. pertussis (lxlO9CFU / mL). Mice were euthanized 4 h or 24 h after administration and PT was quantified in lung homogenates by ELISA.

[0430] Quantification of TRM cells

[0431] To discriminate circulating from tissue-resident lymphocytes by flow cytometry, mice were injected i.v. with 1.5 pg of PE-conjugated anti-CD45 antibody (30-F11; eBioscience) in 200 pl PBS 10 min prior to euthanasia. Circulating lymphocytes are labelled CD45 i.v.+, while tissueresident lymphocytes are protected from i.v. labelling, are therefore identified as CD45 i.v.-. Mononuclear cell suspensions from lungs or nasal tissue were incubated with LIVE / DEAD Aqua (Invitrogen). The cells were incubated with Fc block (BD Biosciences) (1:50) to block IgG Fc receptors followed by a surface staining with fluorochrome-conjugated anti-mouse antibodies specific for CD69 (cloneH1.2F3), CD8 (clone53-6.7), CD3 (clone 145-2C11), CD4 (clone GK1.5), CD44 (clone IM7), CD62L (cloneMEL-14), CD103 (clone M290), and fixed with 2% paraformaldehyde (PFA, Thermo Fisher Scientific). TRM cells were defined through lack of in vivo labelling with anti-CD45. Cells that were CD45i.v. CD44+CD62L- and express CD69, with or without CD103 were considered to be TRM cells.

[0432] For detection of cytokine-producing B. pertussis or S. aureus-specific T cells, mononuclear lung cells were stimulated for 16 hours with heat-killed B. pertussis (HKBp) and heat-killed S. aureus (HKSa) (105CFU / mL), respectively, followed by anti-CD28 and anti-CD49d (lpg / mL; BD Biosciences). Brefeldin A (5pg / ml) was added for the final 4 hours of culture. The cells were fixed, permeabilized and stained using the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions using anti-mouse antibodies specific for IFN-y (XMG1.2) or IL-17 (clone TC11- 18H10). Flow cytometric analysis was performed on an Aurora, and data were acquired using SpectroFlo. Data were analysed using Flowjo software (Tree Star).

[0433] Antigen-specific T cell responses

[0434] Spleens were aseptically removed from immunized mice and mashed through a 70 pm strainer. Spleen cells were cultured at a concentration of lxlO6cells / well in complete RPMI 1640 in the presence of heat-killed B. pertussis (HKBp; lxlO7CFUs / ml), heat-killed S. aureus (HKSa; lxlO7CFU / ml) or with complete RPMI-1640 as a negative control. After 3 days, cell supernatants were harvested, and IL-17 concentrations were quantified using DuoSet ELISA kits (R&D Systems) and IFN-y concentrations were assessed using BD Pharmingen ELISA antibodies according to manufacturers’ protocols and were read using VersaMax microplate reader (Molecular Devices).

[0435] Dendritic cell (DC) stimulation

[0436] Murine bone marrow-derived DCs were prepared from C57BL / 6 mice. DCs (lx 106) in RPMI 1640 medium (without penicillin and streptomycin) were cultured in 96-well U-bottom plates with AIBP vaccine or wP vaccine at concentration equivalent to 2 xlO6bacteria / ml for 24 hours at 37°C in a 5% CO2 incubator. MHCII and co -stimulatory molecule expression was evaluated using flow cytometric analysis. Supernatants were collected from cell cultures for quantification of IL-1 p, IL-6, IL-12p70 and IL-23 by ELISA.

[0437] Activation of / ?, pertussis-specific Thl / Thl7 cells in vitro

[0438] CD4 T cells were enriched from spleens of convalescent mice. This cell preparation contained more than 90% CD4 T cells, with residual macrophages and DCs, which act as antigen- presenting cells (APCs). The enriched CD4 T cells were cultured (2.5 x 106 / ml) with AIBP or the wP vaccine (at concentrations equivalent to 1 xlO7bacteria / ml). After 3 days of culture, the concentrations of IL-17 in supernatants were quantified by ELISA. Alternatively, CD4 T cells were MACS purified from spleens of convalescent mice, and co-cultured (1 x 105 / ml) with increasing concentrations of AIBP or HKBp in the presence of APCs (irradiated spleen cells 2 x 106 / ml). After 3 days of culture, the concentrations of IL-17 and IFN-y in supernatants were quantified by ELISA.

[0439] In vivo treatment with antibodies

[0440] For IL-17 neutralization, mice were injected i.p. with anti-IL-17 antibody (17F3; BioXcell) at 300 pg / mouse. For depletion of CD4 T cells, mice were treated with anti-CD4 antibody (YTS177; BioXcell), 200 pg / mouse i.p. and 100 pg / mouse i.n. (15 pl per nare under anaesthesia) simultaneously 1 day prior to infection and every 3 days after challenge. A corresponding isotype antibody (Rat IgG2b, K; BioXcell) was used as a control. Statistical analysis.

[0441] Statistical analyses were performed using Graph-Pad Prism 9.0 Software. Data were presented as mean ± SEM. Data were analyzed by one-way ANOVAs or two-way ANOVAs followed by post hoc Tukey’s test for multiple comparisons. P values <0.05 were considered significant.

[0442] Results

[0443] Distinct effects of fluoroquinolone antibiotics compared with other antibiotic on the structure of both Gram-negative and Gram-positive bacteria.

[0444] Treatment of Bordetella pertussis in liquid culture with 0.25 to 0.75 mg / ml of levofloxacin resulted in a dose dependant reduction in bacterial growth (Figure 28A). No live bacteria were detected 24 hours after treatment with the higher dose of 0.75 mg / ml of levofloxacin. Furthermore, treatment with levofloxacin at 1, 2 or 3 mg / ml completely inactivated the bacteria; no live bacteria could be detected after 3 hours of culture (Figure 28B). Treatment ofB. pertussis in liquid culture with 25, 50 and 100 pg / ml of chloramphenicol also resulted in a dose dependant reduction in bacterial growth (Figure 27C). Treatment with the higher dose of chloramphenicol (100 pg / ml] completely inactivated the bacteria by 3 hours of culture (Figure 28C).

[0445] Treatment of Staphylococcus aureus in liquid culture with 0.1-0.5 mg / ml ciprofloxacin resulted in a dose dependant reduction in bacterial growth (Figure 29A). Furthermore, treatment with ciprofloxacin at 1, 2 or 3 mg / ml completely inactivated the bacteria; no live bacteria could be detected after 3 hours of culture (Figure 28B). Levofloxacin, another fluoroquinolone antibiotic, had a very similar inactivating effect to ciprofloxacin on the in vitro growth ofS. aureus (Figure 28 C and D).

[0446] Morphology studies by microscopy on B. pertussis 24 hours after antibiotic inactivation revealed intact, and sometimes elongated bacteria, after treatment with levofloxacin (Figure 30A). In contrast, chloramphenicol-treated B. pertussis (Figure 30B] or heat-treated (Figure 30C] had lost their structural integrity compared with live untreated B. pertussis (Figure 30D). The morphology of the chloramphenicol-treated B. pertussis is consistent with lysis of the bacteria by chloramphenicol.

[0447] Morphology studies by microscopy on Escherichia coli 24 hours after antibiotic inactivation revealed intact and elongated bacteria after treatment with levo floxacin (Figure 31 A) or ciprofloxacin (Figure 31B). In contrast, E. coli treated with kanamycin (Figure 31C) or streptomycin (Figure 3 ID) had lost their structural integrity compared with live untreated E. coli (Figure 31E). The morphology of the kanamycin- and streptomycin-treated E. coli is consistent with lysis of the bacteria by these antibiotics.

[0448] Morphology studies by microscopy on S. aureus 24 hours after antibiotic inactivation revealed intact bacteria after treatment with levofloxacin (Figure 32A) or ciprofloxacin (Figure 32B). In contrast, S. aureus treated with penicillin (Figure 32C) or streptomycin (Figure 32D) had lost their structural integrity compared with live untreated S. aureus (Figure 32E). The morphology of the penicillin- and streptomycin-treated S. aureus is consistent with lysis of the bacteria by these antibiotics.

[0449] Bordetella pertussis and Staphylococcus aureus inactivated with fluoroquinolone antibiotics, but not with other antibiotics, are highly immunogenic in vivo, inducing antigen-specific T cells in the spleen and respiratory tract when delivered by the aerosol route to mice.

[0450] The immunogenicity of candidate vaccine preparations prepared by treating Gram-negative or Gram-positive bacteria with fluoroquinolone antibiotics compared with other antibiotics. We also compared 3- and 24-hour treatment protocols for ciprofloxacin. Aerosol immunization of mice with B. pertussis inactivated with ciprofloxacin treated for 3 hours or 24 hours or levofloxacin for 3 hours resulted in expansion of CD4 T cells and CD4 tissue resident memory T (TRM) cells in the lungs of immunized mice (Figure 33 A and B). In contrast, the number of CD4 T cells and CD4 TRM cells in mice immunized with chloramphenicol-inactivated B. pertussis was at background levels, similar to that observed in mice immunized with PBS (Figure 33 A and B).

[0451] Assessment of antigen-specific T cells in the lungs revealed that aerosol immunization of mice with fluoroquinolone antibiotics generated B. pertussis-specific CD4 T cells in the lungs that secreted IFN-y and IL-17, whereas immunization with chloramphenicol-inactivated B. pertussis did not induce B. pertussis T cells (Figure 33 C and D). The T cell responses were not significantly different in mice immunized with the B. pertussis treated with ciprofloxacin for 4 hours compared with 24 hours.

[0452] Assessment of T cell responses in the nose revealed that aerosol immunization of mice with B. pertussis inactivated with ciprofloxacin (3h or 24h) or levo floxacin (3h) resulted in expansion of CD4 T cells and CD4 tissue resident memory T (TRM) cells in the nasal tissue (Figure 34A and B). In contrast, the number of CD4 T cells and CD4 TRM cells in mice immunized with chloramphenicol-inactivated B. pertussis was close to background levels, not significantly different to that observed in mice immunized with PBS (Figure 34 A and B).

[0453] Assessment of antigen-specific T cells in the spleen revealed that aerosol immunization of mice with the B. pertussis inactivated with fluoroquinolone antibiotics, ciprofloxacin (3h or 24h) or levofloxacin (3h) resulted in the induction B. pertussis-specific IFN-y and IL-17, whereas immunization with chloramphenicol-inactivated B. pertussis did not induce B. pertussis-specific cytokine production (Figure 35A and B). B. pertussis-specific IL-17 and IFN- y production by spleen cells was significantly stronger in mice immunized with B. pertussis that had been treated with ciprofloxacin for 3 hours compared with mice immunized with B. pertussis treated with ciprofloxacin for 24 hours.

[0454] Assessment of the immunogenicity of S. aureus treated with levofloxacin revealed that aerosol immunization of mice with S. aureus inactivated with levofloxacin for 3 hours resulted in significant expansion of CD4 T cells and CD4 TRM cells in the nasal tissue (Figure 36 A and B) and lungs (Figure 36C and D). Assessment of antigen-specific T cells in the lungs revealed that aerosol immunization of mice with levofloxacin-treated S. aureus generated IFN-y- and IL-17-secretingS. aureus-specific CD4 T cells in the lungs (Figure 36 E and F).

[0455] Assessment of antigen-specific T cells in the spleen revealed that aerosol immunization of mice with the S. aureus inactivated with levofloxacin induced S. aureus-specific IFN-y and IL- 17, (Figure 37A and B). S. aureus-specific IL-17 and IFN-y production by spleen cells was significantly stronger in mice immunized with B. pertussis that had been treated with ciprofloxacin for 3 hours compared with mice immunized with PBS (Figure 37A and B).

[0456] Further testing of AIBP vaccine safety

[0457] We assessed the potential safety of the AIBP vaccine. No live B. pertussis could be detected in the lungs or nose after aerosol delivery of the AIBP vaccine to mice, showing that AIBP do not colonize the respiratory tract, whereas significant CFU were detected in mice given an aerosol of live B. pertussis (Figure 38A). We also assessed in vivo production of pertussis toxin (PT) a major contributor to pertussis disease. We detected significant concentrations of PT in the lungs of mice following aerosol of live B. pertussis, but not following aerosol of AIBP (Figure 38B). These findings, together with the lack of systemic pro-inflammatory cytokine production, demonstrate that the AIBP vaccine is unlikely to be rectogenic in vivo. The AIBP vaccine is significantly more effective than the wP vaccine in activating antigen presenting cells (APC).

[0458] Dendritic cells (DCs) play a central role as APCs in priming naive T cells. We assessed the effect of the AIBP vaccine on DC maturation. The AIBP vaccine enhanced expression of MHCII, CD40 and CD80 on bone-marrow derived DCs and this was significantly greater than that induced with the wP vaccine (Figure. 39A). The AIBP vaccine promoted production of the T cell polarizing cytokines IL-1 p, IL-12p70 and IL-23, which was significantly greater than that induced with the wP vaccine (Figure 39B).

[0459] Finally, we assessed the ability of the AIBP vaccine to stimulate B. pertussis-specific T cell responses in vitro. B. pertussis-specific CD4 T cells, with residual APC, from spleens of convalescent mice, secreted IL-17 when cultured with AIBP and this was significantly greater than observed following culture with the wP vaccine (Figure 39C).

[0460] We also assessed the ability of the AIBP vaccine to stimulate B. pertussis-specific Thl and Thl7 cells through activation of APCs. B. pertussis-specific CD4 T cells, purified from spleens of convalescent mice, secreted IL-17 and IFN-y when cultured with AIBP in the presence of APC (irradiated spleen cells) and this was significantly greater than that induced with heat- killed B. pertussis (HKBp), especially at low antigen concentrations (Figure 40). Collectively, our findings demonstrate that the AIBP vaccine induces DC maturation and production of T cell polarizing cytokines and activates APC to drive Thl and Thl7 responses.

[0461] Mechanism of protective immunity induced with the AIBP vaccine.

[0462] Although antibodies play a role in preventing infection of the lungs in mice[l, 2] and maternal antibodies protect against pertussis disease in human infants[3], studies in mice have shown IL-17-secreting T cells are required for clearance of B. pertussis from the nasal tract[4]. Studies in mice, baboons and humans have shown that current aP vaccines selectively induce Th2 cells and antibody responses, but not respiratory TRM cells, and consequently fail to prevent nasal colonization with B. pertussis[l, 5-7], We examined the possible role of IL-17- secreting CD4 T cells by depleting CD4 cells or neutralizing IL-17 before and after challenge with B. pertussis in mice immunized with one dose of the AIBP vaccine. Protection induced with a single dose of the AIBP vaccine was completely abrogated after depletion of CD4 T cells or neutralization of IL-17; the CFU counts in the anti-CD4 depleted mice were similar to those in non-immunized control mice (Figure 41A).

[0463] Flow cytometry analysis demonstrated that recruitment of CD4 T cells to the lungs and nose in mice immunized with the AIBP vaccine was abrogated in mice treated with the anti-CD4 antibody and was also reduced in mice treated with the anti-IL-17 antibody (Figure 41B). IL- 17 is known to recruit neutrophils, especially Siglec-F+neutrophils to the respiratory tissue of B. pertussis infected mice. Here we found enhanced recruitment of Siglec-F+neutrophils to the lungs and nasal tissue post B. pertussis challenge of mice immunized with the AIBP and this was reversed in mice treated with anti-IL-17 and significantly reduced in mice treated with anti-CD4 (Figure 41C). Our findings demonstrate that the AIBP vaccine mediates sterilizing immunity largely via induction of IL-17-secreting CD4 TRM cells that promote recruitment of Siglec-F+neutrophils to the respiratory tract.

[0464] Discussion

[0465] Our findings demonstrate that candidate vaccines prepared by inactivation of Gram-negative or Gram-positive bacteria with fluoroquinolone antibiotics, but not with other antibiotics, are highly immunogenic when delivered to mice via the respiratory tract. Aerosol administration of B. pertussis or S. aureus treated with either ciprofloxacin or levofloxacin induce potent antigen-specific IFN-y and IL-17-secreting T cell responses in the periphery and in the respiratory tract. These candidate vaccines also induced CD4 TRM cells in the lung and nasal tissue. Respiratory TRM cells play a crucial role in protective immunity against B. pertussis and S. aureus. IL-17-secreting CD4 T cells play a crucial role in protective immunity to B. pertussis and S. aureus in the nasal mucosa [8, 9], In contrast, immunization with B. pertussis treated with chloramphenicol did not generate B. pertussis specific T cell responses, suggesting that the treatment with fluoroquinolone antibiotics creates highly immunogenic bacteria.

[0466] The superior immunogenicity of bacteria treated with fluoroquinolone antibiotics compared with other antibiotic treatment reflects the difference in the structure of the bacteria observed post antibiotic treatment. Treatment of the Gram-negative bacteria, B. pertussis and E. coli, or the Gram-positive S. aureus with the fluoroquinolone antibiotics, ciprofloxacin or levofloxacin did not result in bacterial lysis. The bacteria were intact, and in some cases elongated, consistent with the mode of action of this family of antibiotics which inhibit DNA synthesis, but do not disrupt the bacterial cell wall. In contrast, bacteria treated with chloramphenicol, kanamycin, penicillin or streptomycin had lost their characteristic morphology and appeared to be lysed. This may explain the superior immunogenicity of the fluoroquinolone-treated bacteria.

[0467] Overall, our data reveals that compared with other antibiotics, treatment of Gram-positive or Gram-negative bacteria with fluoroquinolone antibiotics is an ideal approach to bacterial inactivation to create a vaccine that is highly immunogenic. The fluoroquinolone-inactivated bacteria are still intact and sometime enlarged 24 hours after treatment, whereas bacteria treated with other antibiotics, including chloramphenicol, kanamycin, penicillin or streptomycin are lysed within 24 hours. Whole bacteria, especially if enlarged, are likely to be more potent immunogens than lysed bacteria, because they are more likely to be taken up by, and activate, antigen presenting cells and thereby promote stronger T cell responses. This theory is supported by our demonstration that bacteria treated with fluoroquinolone, but not other antibiotics, are highly immunogenic, inducing potent antigen-specific Thl and Thl7 and TRM cells in the respiratory tract.

[0468] Strong T cell responses were observed when the bacteria were treated for 3 hours with the fluoroquinolone antibiotics. Bacteria treated with fluoroquinolone antibiotics for 24 hours were also immunogenic, but the responses were in some cases weaker than those observed for bacteria treated with fluoroquinolone antibiotics for 3 hours. This suggests that there is window after treatment with fluoroquinolone antibiotics when the bacteria are most immunogenic and this may reflect their structure, which is lost when the bacteria are treated with other antibiotics.

[0469] In terms of safety, aerosol delivery of the AIBP vaccine did not promote systemic production of proinflammatory cytokines, observed with a parenterally-delivered wP vaccine, which has been linked to adverse events. Furthermore, it did not colonize in lungs or nose or secrete PT in immunized mice.

[0470] We found that the AIBP vaccine has very distinct characteristics and properties to wP vaccines. wP vaccines are prepared by inactivating B. pertussis with aldehydes, usually formaldehyde, which cross links proteins by forming covalent bonds between different amino acid residues. This can adversely affect conformational antibody epitopes and alter protein degradation by endo-lysosomal proteases, thereby affecting antigen processing and T cell activation

[0010] . Furthermore, conformational changes to proteins, such as those induced by aldehyde (i.e. chemical) treatment can hamper antigen processing and T cell activation[ll]. This may affect the immunogenicity and protective efficacy of the killed bacteria in the wP vaccines. In contrast, in the bacterial antigens are not modified in the AIBP vaccine and our data demonstrate that the AIBP vaccine is more effective than wP vaccine at stimulating APC and thereby promoting Thl and Thl7 responses. Furthermore, the AIBP vaccines induces significantly greater accumulation of Thl and Thl7-type TRM cells in the respiratory tissue and confers better protection against B. pertussis infection of the nasal cavity. The AIBP vaccine induced DC maturation and production of the T cell polarizing cytokines IL-ip, IL-12p70 and IL-23, which was significantly greater than that induced with the wP vaccine. This is consistent with the highly effective priming of IL-17 and IFN-y-secreting respiratory CD4 TRM cells. Although two doses were required to induce potent serum IgG and mucosal IgA, a single dose of the AIBP vaccine induced respiratory TRM cells and conferred complete protection against lung infection and high level of protection against B. pertussis infection of the nasal tract. This protection was significantly attenuated following depletion of CD4 T cells or neutralization of IL-17, suggesting that IL-17-producing CD4 TRM cells are major mediators of protective immunity induced with the AIBP vaccine.

[0471] As well as the superior immunogenicity and protective efficacy, another significant advantages of the AIBP vaccine over aP vaccine the ease and cost of production. Furthermore, respiratory administration of the vaccine delivery with a simple nebulizer would allow needle-free and pain-free delivery, improving compliance and vaccine uptake. Because of the multiple antigens expressed by the AIBP vaccine, it has the capacity protect against circulating strains with mutations or deletions in B. pertussis antigens present in the aP vaccine. It also has the versatility to be adapted to cope with strains of B. pertussis that may emerge in the future. Therefore, this new technology has considerable potential for the development of a more effective vaccine against infection with B. pertussis and other respiratory bacteria in human.

[0472] Our unexpected discovery reveals that transient treatment of Gram-positive or Gramnegative bacteria with fluoroquinolone antibiotics is an ideal vaccine platform approach for the development of potent vaccines against a range of infectious diseases. While the fluoroquinolone-treated bacteria are highly immunogenic when delivered by aerosol or intranasal routes, this vaccine platform could also be used to prepare vaccines against infectious diseases at other mucosal surfaces, such as the gastrointestinal tract, which could be facilitated by oral delivery of the vaccine. Additionally, this vaccine platform could also be used to prepare vaccines against infectious diseases of the skin or bloodstream, caused by S. aureus for example, which could be facilitated by parenteral (subcutaneous, intradermal, transdermal or even intramuscular) delivery of the vaccine. References

[0473] 1 Mills, K. H., Ryan, M., Ryan, E. and Mahon, B. P., A murine model in which protection correlates with pertussis vaccine efficacy in children reveals complementary roles for humoral and cell-mediated immunity in protection against Bordetella pertussis. Infect Immun 1998. 66: 594-602.

[0474] 2 Callender, M. and Harvill, E. T., Maternal vaccination: shaping the neonatal response to pertussis. Front Immunol 2023. 14: 1210580.

[0475] 3 Van Rie, A., Wendelboe, A. M. and Englund, J. A., Role of maternal pertussis antibodies in infants. Pediatr Infect Dis J 2005. 24: S62-65.

[0476] 4 Borkner, L., Curham, L. M., Wilk, M. M., Moran, B. and Mills, K. H. G., IL-17 mediates protective immunity against nasal infection with Bordetella pertussis by mobilizing neutrophils, especially Siglec-F(+] neutrophils. Mucosal Immunol 2021. 14: 1183-1202.

[0477] 5 Wilk, M. M., Borkner, L., Misiak, A., Curham, L., Allen, A. C. and Mills, K. H. G., Immunization with whole cell but not acellular pertussis vaccines primes CD4 T(RM) cells that sustain protective immunity against nasal colonization with Bordetella pertussis. Emerg Microbes Infect 2019. 8: 169-185.

[0478] 6 Warfel, J. M., Zimmerman, L. I. and Merkel, T. J., Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model. Proc Natl Acad Sci USA 2014. Ill: 787-792.

[0479] 7 McCarthy, K. N., Hone, S., McLoughlin, R. M. and Mills, K. H. G., IL-17 and IFN-y- producing Respiratory Tissue-Resident Memory CD4 T Cells Persist for Decades in Adults Immunized as Children With Whole-Cell Pertussis Vaccines. J Infect Dis 2024. 230: e518- e523.

[0480] 8 Murphy, A. G., O'Keeffe, K. M., Lalor, S. J., Maher, B. M., Mills, K. H. and McLoughlin, R. M., Correction: Staphylococcus aureus Infection of mice expands a population of memory y8 T cells that are protective against subsequent infection. J Imm unol 2015. 194: 4588.

[0481] 9 Archer, N. K., Harro, J. M. and Shirtliff, M. E., Clearance of Staphylococcus aureus nasal carriage is T cell dependent and mediated through interleukin-17A expression and neutrophil influx. Infect Immun 2013. 81: 2070-2075.

[0482] 10 Michiels, T. J. M., Meiring, H. D., Jiskoot, W., Kersten, G. F. A. and Metz, B., Formaldehyde treatment of proteins enhances proteolytic degradation by the endo-lysosomal protease cathepsin S. Sci Rep 2020. 10: 11535.

[0483] 11 Mills, K. H., Processing of viral antigens and presentation to class Il-restricted T cells. Immunol Today 1986. 7: 260-263. The invention will now be described by the following non-limiting sequentially numbered statements:

[0484] 1. A vaccine composition comprising bacteria inactivated by an antibiotic or an antibacterial compound for use in the prevention of infection with the bacteria and / or in the treatment and / or prophylaxis of a disease associated with the bacteria.

[0485] 2. The vaccine composition of statement 1, wherein the vaccine composition prevents infection of the nose and lungs with the bacteria.

[0486] 3. The vaccine composition of statements 1 or 2, wherein the bacteria is a gram negative or a gram positive bacteria; optionally wherein the bacteria is a bacteria that causes respiratory tract infections or a disease of the respiratory tract.

[0487] 4. The vaccine composition of any preceding statement, wherein the bacteria is Bordetella pertussis and the disease associated with the bacteria is pertussis disease.

[0488] 5. The vaccine composition of any preceding statement, wherein the vaccine composition is administered by aerosol delivery.

[0489] 6. The vaccine composition of any preceding statement, wherein the vaccine composition is formulated for administration at a mucosal site; optionally wherein the vaccine composition is formulated for intranasal administration.

[0490] 7. The vaccine composition of any preceding statement, wherein the vaccine composition is administered by aerosol delivery using a nebuliser from a culture at lxlO5- lxlO9CFU / ml or by intranasal administration at a dose of 1X105-1X109CFU in 0.1-1.0 ml PBS.

[0491] 8. An aerosol-delivered antibiotic-inactivated Bordetella pertussis (AIBP] vaccine or a Bordetella pertussis vaccine wherein the Bordetella pertussis has been inactivated with an antibacterial compound for use in preventing infection with Bordetella pertussis and / or in the treatment and / or prophylaxis of pertussis disease. 9. The vaccine of statement 8, wherein the vaccine composition prevents infection of the nose and lungs with B. pertussis.

[0492] 10. The vaccine of statements 8 or 9, wherein the vaccine composition is formulated for administration at a mucosal site; optionally wherein the vaccine composition is formulated for intranasal administration.

[0493] 11. The vaccine of any of statements 8-10, wherein the vaccine composition is administered by aerosol delivery using a nebuliser from a culture at 1X105-1X109CFU / ml or by intranasal administration at a dose of 1X105-1X109CFU in 0.1-1.0 ml PBS.

[0494] 12. The vaccine of any of statements 8-11, wherein the vaccine is delivered using an aerosol delivery device; optionally wherein the aerosol delivery device is a basic pressurized metered-dose inhaler, a nebulizer, a valved holder chambers inhaler, a dry powder inhaler, a soft mist inhaler, a nasal spray device, a nasal sprayer with syringe or a smart inhaler.

[0495] 13. A vaccine for use in vaccinating a subject against infection with a bacteria and / or a disease associated with the bacteria, wherein the vaccine comprises the bacteria inactivated with an antibiotic or an antibacterial compound.

[0496] 14. A composition comprising a bacteria inactivated with an antibiotic or an antibacterial compound for use in mediating an immune system in a subject and for preventing infection with the bacteria and / or in the treatment and / or prophylaxis of a disease associated with the bacteria.

[0497] 15. The vaccine of statement 13 or the composition of statement 14, wherein the bacteria is a gram negative or a gram positive bacteria; optionally wherein the bacteria is a bacteria that causes respiratory tract infections or a disease of the respiratory tract.

Claims

CLAIMS1. A vaccine composition comprising bacteria inactivated by a fluoroquinolone antibiotic.

2. The vaccine composition of claim 1, wherein the bacteria inactivated by the fluoroquinolone antibiotic is not lysed.

3. The vaccine composition of claim 1 or 2, wherein the immunogenicity of the bacteria inactivated by the fluoroquinolone antibiotic is maintained and / or enhanced compared to bacteria inactivated by heat treatment or chemical treatment.

4. The vaccine composition of claims 1 to 3, wherein the bacteria is a gram- negative or a gram-positive bacteria, including acid-fast bacteria.

5. The vaccine composition of any preceding claim, wherein the bacteria causes respiratory tract infections or a disease of the respiratory tract.

6. The vaccine composition of any preceding claim, wherein the bacteria is selected from one or more of Bordetella pertussis, Staphylococcus aureus, Burkholderia pseudomallei, Chlamydophila pneumoniae, Corynebacterium diphtheriae, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, Streptococcus aures, Streptococcus pyogenes, Escherichia Coli, Pseudomonas aeruginosa, Coxiella burnetiid, Legionella pneumophila, Mycobacterium tuberculosis and non-tuberculous mycobacteria.

7. The vaccine composition of any preceding claim, wherein the bacteria is Bordetella pertussis and the disease associated with the bacteria is pertussis disease.

8. The vaccine composition of any preceding claims, wherein the vaccine composition prevents infection of the nose and lungs with the bacteria.

9. The vaccine composition of any preceding claims, wherein the vaccine composition comprising bacteria inactivated by the fluoroquinolone antibiotic provides a greater immune response, in terms of activating antigen presenting cells and inducing protective T cellresponses in lung and nasal tissue compared to bacteria inactivated by heat treatment or chemical treatment.

10. The vaccine composition of any preceding claims, wherein the vaccine composition comprising bacteria inactivated by the fluoroquinolone antibiotic provides a greater immune response in terms of inducing Thl7 response in lung and nasal tissue compared to bacteria inactivated by heat treatment or chemical treatment.

11. The vaccine composition of any preceding claim, wherein the vaccine composition is administered by aerosol delivery.

12. The vaccine composition of any preceding claim, wherein the vaccine composition is formulated for administration at a mucosal site; optionally wherein the vaccine composition is formulated for intranasal, aerosol, pulmonary or oral delivery; preferably wherein the vaccine composition is formulated for intranasal administration.

13. The vaccine composition of any preceding claim, wherein the vaccine composition is administered by aerosol delivery using a nebuliser from a culture at 1X105-1X109CFU / ml; or by intranasal administration at a dose of 1X105-1X109CFU in 0.1-1.0 ml PBS.

14. The vaccine composition of any claims 1 to 10, wherein the vaccine composition is formulated for parenteral administration, optionally wherein the vaccine composition is formulated for injection; still optionally wherein the vaccine composition is formulated for sub-cutaneous, trans-dermal or intradermal delivery.

15. The vaccine composition of claim 14, wherein the bacteria is Staphylococcus aureus and the disease associated with the bacteria is a skin infection and / or bloodstream infection.

16. An aerosol-delivered fluoroquinolone antibiotic-inactivated Bordetella pertussis (Al BP) vaccine,wherein the vaccine prevents infection of the nose and lungs with B. pertussis.

17. The vaccine of claim 16, wherein the vaccine is formulated for administration at a mucosal site; optionally wherein the vaccine is formulated for intranasal administration.

18. The vaccine of claims 16 or 17, wherein the vaccine composition is administered by aerosol delivery using a nebuliser from a culture at 1X105-1X109CFU / ml; or by intranasal administration at a dose of 1X105-1X109CFU in 0.1-1.0 ml PBS.

19. The vaccine of claims 16 to 18, wherein the vaccine is delivered using an aerosol delivery device; optionally wherein the aerosol delivery device is a basic pressurized metered-dose inhaler, a nebulizer, a valved holder chambers inhaler, a dry powder inhaler, a soft mist inhaler, a nasal spray device, a nasal sprayer with syringe or a smart inhaler.

20. The vaccine composition of claims 1 to 15 or the vaccine of claims 16 to 19, comprising ciprofloxacin, gemifloxacin, levo floxacin, moxifloxacin, delafloxacin, norfloxacin and / or ofloxacin-inactivated bacteria.

21. The vaccine composition of claims 1 to 13 or the vaccine of claims 16 to 20, comprising ciprofloxacin, gemifloxacin, levo floxacin, moxifloxacin, delafloxacin, norfloxacin and / or ofloxacin-inactivated B. pertussis.

22. The vaccine composition of claims 1 to 13 or the vaccine of claims 16 to 20, comprising ciprofloxacin-inactivated B. pertussis.

23. The vaccine composition of claims 1 to 15 or the vaccine of claims 16 to 22, which comprises or does not comprise an adjuvant, such as aluminium salt adjuvant.

24. The vaccine composition of claims 1 to 15 and 20 to 23, or the vaccine of claims 16 to 23, for use in inducing an immune response in a subject against the bacteria.

25. The vaccine composition of claims 1 to 15 and 20 to 23, or the vaccine of claims 16 to 23, for use in preventing of infection with the bacteria and / or in the treatment and / or prophylaxis of a disease associated with the bacteria.

26. The vaccine composition or vaccine for use according to claims 24 or 25, wherein the bacteria causes respiratory tract infections or a disease of the respiratory tract.

27. The vaccine composition or vaccine for use according to claims 24 or 25, in preventing infection with Bordetella pertussis and / or in the treatment and / or prophylaxis of pertussis disease.

28. The vaccine composition or vaccine for use according to claims 24 or 25, wherein the bacteria causes a skin disease or infection, and / or a bloodstream infection.

29. The vaccine composition or vaccine for use according to claims 24, 25 or 28, in preventing infection with Staphylococcus aureus and / or in the treatment and / or prophylaxis of a skin disease or infection and / or a bloodstream infection.

30. A method for preventing infection with bacteria and / or for the treatment and / or prophylaxis of a disease associated with the bacteria, said method comprising the step of:(i) administering to a subject in need thereof a therapeutically effective amount of a vaccine composition comprising the bacteria inactivated with a fluoroquinolone antibiotic.

31. A method of inducing an immune response in a subject against a bacteria, said method comprising the steps of:(i) providing a vaccine composition comprising a bacteria inactivated with a fluoroquinolone antibiotic, and(if) administering the vaccine composition to the subject in a therapeutically effective or prophylactically effective amount.

32. A method for method for preventing infection with bacteria and / or for the treatment and / or prophylaxis of a disease associated with the bacteria; comprising administering to the subject a therapeutically effective amount of the vaccine composition of claims 1 to 15 or the vaccine of claims 16 to 23.

33. The method of claims 30 to 32, wherein the administering induces a protective immune response in the subject, and wherein the immune response comprises an immune response against said bacteria.

34. The method of claims 30 to 33, wherein the administering is selected from the group consisting of subcutaneous delivery, transdermal or intradermal delivery, transcutaneous delivery, intradermal delivery, subdermal delivery, intramuscular delivery, peroral delivery, oral delivery, intranasal delivery, buccal delivery, sublingual delivery, intraperitoneal delivery, intravaginal delivery, anal delivery and intracranial delivery.

35. A method for making a fluoroquinolone antibiotic inactivated bacterial vaccine, comprising:(a) culturing bacteria from frozen stocks or clinical isolates;(b) treating the cultured bacteria from (a) with an effective amount of fluoroquinolone antibiotic over an effective time period such that no live bacterial colony forming units (CFU) are present; and(c) washing the treated bacteria from (b) free of fluoroquinolone antibiotic to result in a fluoroquinolone inactivated bacterial vaccine.

36. A method for inactivating a B. pertussis isolate, comprising:(a) culturing B. pertussis bacteria from frozen stocks of B. pertussis Tohama-1 derivatives, or from B. pertussis clinical isolates;(b) treating the cultured B. pertussis bacteria from (a) with an effective amount of a fluoroquinolone antibiotic over a time period of approximately 1 to 24 hours such that no live B. pertussis CFU are present; and(c) washing the treated B. pertussis bacteria from (b) free of fluoroquinolone antibiotic.

37. A method for making a fluoroquinolone antibiotic inactivated B. pertussis (AIBPj vaccine, comprising:(a) culturing B. pertussis bacteria from frozen stocks of B. pertussis Tohama-1 derivatives, or from B. pertussis clinical isolates;(b) treating the cultured B. pertussis bacteria from (a) with an effective amount of fluoroquinolone antibiotic over a time period of approximately 1 to 24 hours such that no live B. pertussis CFU are present; and(c) washing the treated B. pertussis from (b) free of the fluoroquinolone antibiotic to result in a fluoroquinolone inactivated B. pertussis vaccine.

38. The method of claims 35 to 37, wherein the bacterial culture has a CFU of approximately lxlO9.

39. The method of claims 35 to 38, where the effective amount of fluoroquinolone antibiotic is from 0.01 to 3mg / ml.

40. The method of claims 35 to 39, wherein the time period is from approximately 2-24 hours, preferably approximately 3 hours.

41. The method of claims 35 to 40, wherein the fluoroquinolone antibiotic is selected from one or more of ciprofloxacin, gemifloxacin, levofloxacin, moxifloxacin, delafloxacin, norfloxacin and ofloxacin; preferably ciprofloxacin or levofloxacin.

42. The method of claims 35 to 41, wherein the method does not comprise a heat inactivation step or a chemical (aldehyde) inactivation step either before or after the antibiotic or fluoroquinolone treatment step.

43. The vaccine composition of claims 1 to 15 or the vaccine composition of claims 16 to 23, for use in primary vaccination or as a booster.

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